Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals' blood cells in combination with CpG-DNA.

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Effect of cigarette smoke on mucosal vaccine response with activation of plasmacytoid dendritic cells: The outcomes of in vivo and in vitro experiments.

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.

Evaluation of a booster tuberculosis vaccine containing mycobacterial DNA-binding protein 1 and CpG oligodeoxynucleotide G9.1 using a Guinea pig model that elicits immunity to Bacillus Calmette-Guérin.

Tuberculosis is a major threat to global health and its increased incidence in adolescents as well as onset in the elderly presents a serious problem. One strategy to control tuberculosis involves taking advantage of Bacillus Calmette-Guérin's (BCG) superior effects on childhood tuberculosis. Accordingly, here we aimed to develop a booster vaccine for adults who received the BCG vaccine during early childhood. Therefore, we first devised a system to assess the efficacy of a candidate booster vaccine. Specifically, variant strain BCG-II, a minor component of BCG-Tokyo strain, which elicits weak immunity, was administered to guinea pigs. Vaccine-induced immunity and protection against Mycobacterium tuberculosis (Mtb) infection were evaluated using skin delayed-type hypersensitivity (DTH) and Mtb colony forming unit counts in organs, respectively. Candidate booster vaccine containing the mycobacterial DNA-binding protein 1 (MDP1) as antigen and CpG oligodeoxynucleotide G9.1 as adjuvant increased T-bet expression and IFN-γ production in human peripheral blood mononuclear cells. Intradermal administration of MDP1 or MDP1 and G9.1 to unimmunized guinea pigs produced DTH on MDP1-inoculated skin. Boosting BCG-II-primed guinea pigs with this protocol effectively enhanced DTH against MDP1 and protection against Mtb infection, particularly when combined with G9.1. The candidate vaccine may contribute to efforts to prevent tuberculosis.

Overexpression of neural miRNAs miR-9/9* and miR-124 suppresses differentiation to Müller glia and promotes differentiation to neurons in mouse retina in vivo.

MicroRNAs (miRNAs) are known to regulate gene expression and modulate cellular differentiation. MicroRNA-9/9* (miR-9/9*) and microRNA-124 (miR-124) are highly expressed in the central nervous system. In vivo function of miR-9/9* and miR-124 has been investigated in detail, whereas there remain some discrepancies regarding neural development. To this end, we electroporated miR-9/9*, miR-124 or miR-9/9*/124 expression plasmids into neonatal retinal progenitor cells (RPCs) in vivo and analyzed the fate of electroporated cells. Both miR-9/9* and miR-124 reduced the number of SOX9- and GS-positive cells and increased that of TUBB3-positive cells in the postnatal day 14 retina. No major effects on the proliferation and apoptosis of the electroporated cells were detected at least postnatal day 3. These indicated that miR-9/9* and miR-124 influence the cell fate of glial cells, thereby inducing their differentiation into neurons. Moreover, we found this cell fate modulation was occurred in RPCs indicating high-level expression of miRNA, but not in the low level. Our results strongly suggest that high-level miRNA overexpression is essential for directing cell fate by miR-9/9* and miR-124 interference.

Normal aging hyperactivates innate immunity and reduces the medical efficacy of minocycline in brain injury.

Symptoms of many neurodegenerative diseases appear later in human life. However, young animal models for penetrating traumatic brain injury (pTBI) have been used to study neurodegenerative diseases and evaluate the efficacy of neuroprotective medicines. Possibly because of this discordance, effective neuroprotective drugs have still not been developed. For patients suffering from pTBI, aging is known to be a significant prognostic factor of mortality. In this study, we aimed to establish a model of aged pTBI animals using Drosophila melanogaster. We successfully generated aged pTBI flies as a new pTBI model showing increased neurodegeneration and higher mortality. To elucidate the mechanism of increased vulnerability in aged pTBI animals, we analyzed the GenBank-deposited transcriptome data of young and aged flies, demonstrating the importance of innate immunity genes for higher mortality in aged pTBI models. We found that in the context of pTBI, normal aging strongly activated the expression of antimicrobial peptide genes and upregulated the nuclear factor-κB gene in the immune deficiency pathway, but not the Toll pathway. Moreover, we found that minocycline increased the survival of young pTBI flies, but not aged pTBI flies. These results suggested that immune system activation under neurodegenerative conditions was involved in normal aging, thereby inhibiting the medicinal efficacy of neuroprotective drugs effective for young flies in aged flies.

Treatment with Lactobacillus Retards the Tumor Growth of Head and Neck Squamous Cell Carcinoma Cells Inoculated in Mice.

Bacteria have been used for more than a century to treat solid tumors. Because solid tumors generate an anaerobic environment, we evaluated the anti-tumor effect of the obligate anaerobe strain KK378, derived from Lactobacillus casei (L. casei), using mice bearing head and neck cancer. Wild-type L. casei is a nonpathogenic bacterium that is commonly used in foods. Moreover, patients with head and neck squamous cell carcinoma often have multiple cancers and cervical lymph node metastasis that can be directly sensed beneath the skin. To establish the animal model bearing head and neck cancer, we inoculated each of human squamous cell carcinoma cell lines, SAS, HSQ89, and HSC2, on the back skin of BALB/cSlc-nu/nu mice. After tumor formation, L. casei KK378 was administered directly into the tumor, and tumor size and serum cytokine levels were analyzed. Mice injected with 108 cfu of L. casei KK378 showed reduction in tumor growth compared with PBS control; especially, the SAS tumor was significantly reduced (p = 0.008). Administered L. casei KK378 was detected in tumor tissues but not in normal tissues (liver, kidney, and lung) of SAS tumor-bearing mice, which was associated with increased blood cytokines (TNF-α, IFN-γ, IL-5, IL-10, and IL-12). Among these cytokines, the serum levels of IFN-γ and TNF-α were significantly increased (p < 0.05). In conclusion, L. casei KK378 infection may suppress tumor growth by inducing the host immune response. Direct injection of Lactobacillus into the tumor could be a potential strategy to treat head and neck squamous cell carcinoma.

Life-Sustaining Treatment Status at the Time of Death in a Japanese Pediatric Intensive Care Unit.

BACKGROUND: Substantial variability exists among countries regarding the modes of death in pediatric intensive care units (PICUs). However, there is limited information on end-of-life care in Japanese PICUs. Thus, this study aimed to elucidate the characteristics of end-of-life care practice for children in a Japanese PICU. METHODS: We examined life-sustaining treatment (LST) status at the time of death based on medical chart reviews from 2010 to 2014. All deaths were classified into 3 groups: limitation of LST (limitation group, death after withholding or withdrawal of LST or a do not attempt resuscitation order), no limitation of LST (no-limitation group, death following failed resuscitation attempts), or brain death (brain death group). RESULTS: Of the 62 patients who died, 44 (71%) had limitation of LST, 18 (29%) had no limitation of LST, and none had brain death. In the limitation group, the length of PICU stay was longer than that in the no-limitation group (13.5 vs 2.5 days; P = .01). The median time to death after the decision to limit LST was 2 days (interquartile range: 1-5.5 days), and 94% of the patients were on mechanical ventilation at the time of death in the limitation group. CONCLUSIONS: Although limiting LST was a common practice in end-of-life care in a Japanese PICU, a severe limitation of LST such as withdrawal from the ventilator was hardly practiced, and a considerable LST was still provided at the time of death.

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery.

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed ß-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st ß/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd ß/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, ß-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.

Hyperoxia causes diffuse alveolar damage through mechanisms involving upregulation of c-Myc/Bax and enhanced production of reactive oxygen species.

BACKGROUND: Hyperoxia is a known cause of diffuse alveolar damage (DAD). We previously reported the transcript profiling of DAD induced by hyperoxia exposure in mouse lungs and showed that the gene expression of myelocytomatosis oncogene (c-Myc) was significantly upregulated whereas that of surfactant-associated protein (SP)-C was downregulated. However, the mechanism underlying hyperoxia-induced DAD is not well understood. METHODS: The hyperoxia-induced changes in SP-A/B/C/D, c-Myc, B-cell chronic lymphocytic leukemia/lymphoma (Bcl)-2, and Bcl-2-associated X protein (Bax) expression in mouse lungs were examined by cDNA microarray analysis. The expression levels of the above mentioned genes, cell viability, caspase activity, and reactive oxygen species (ROS) production were also examined in the human lung adenocarcinoma cell line A549 and mouse fibroblast-like cell line NIH/3T3. RESULTS: Hyperoxia induced a decrease in SP-C/A expression in mouse lungs, and SP-C downregulation was also confirmed in A549 cells. In addition to enhanced c-Myc expression, Bax expression also increased following exposure of the mice to hyperoxia. In vitro analysis showed that expression of these genes is regulated in a cell-type-dependent manner, i.e., upregulation of c-Myc in NIH/3T3 cells and Bax in A549 cells occurred regardless of whether there was a similar decrease in cell viability and increase in caspase-3/7 activation in response to hyperoxia. ROS production and caspase-8 activation were also observed in both cells. CONCLUSIONS: We concluded that hyperoxia induces ROS production and cell death in lung tissues through a cell-type specific mechanism involving the upregulation of c-Myc/Bax, and caspase-8 and -3/7 activation-dependent pathways, thereby leading to the development of DAD.

CpG oligodeoxynucleotides as mucosal adjuvants.

Bacterial DNA comprising palindromic sequences and containing unmethylated CpG is recognized by toll-like receptor 9 of plasmacytoid dendritic cells (pDCs) and induces the production of interferon-α and chemokines, leading to the activation of a Th1 immune response. Therefore, synthetic equivalents of bacterial DNA (CpG oligodeoxynucleotides) have been developed for clinical applications. They are usually phosphorothioated for in vivo use; this approach also leads to adverse effects as reported in mouse models.Mucosal vaccines that induce both mucosal and systemic immunity received substantial attention in recent years. For their development, phosphodiester-linked oligodeoxynucleotides, including the sequence of a palindromic CpG DNA may be advantageous as adjuvants because their target pDCs are present right there, in the mucosa of the vaccination site. In addition, the probability of adverse effects is believed to be low. Here, we review the discovery of such CpG oligodeoxynucleotides and their possible use as mucosal adjuvants.

Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains.

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150 kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a ß-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N'-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.

Crystallization and preliminary X-ray analysis of the catalytic domains of Paenibacillus sp. strain FPU-7 cell-surface-expressed chitinase ChiW.

The polysaccharide chitin is effectively hydrolyzed and utilized as a carbon and nitrogen source by the Gram-positive bacterium Paenibacillus sp. strain FPU-7. ChiW is a unique cell-surface-expressed chitinase among the Paenibacillus sp. strain FPU-7-secreted chitinases. An N-terminally truncated ChiW protein, primarily comprised of the two catalytic domains of the full-length protein, was successfully overexpressed in Escherichia coli, purified as a functional recombinant protein with a molecular mass of approximately 98 kDa and crystallized. Preliminary X-ray analysis showed that the crystal diffracted to 1.93 Šresolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 112.1, b = 128.2, c = 162.6 Å, suggesting the presence of two molecules in an asymmetric unit.

A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H)1 immunity.

BACKGROUND: CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated T(H)1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H)1 and T(H)2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H)1, but not T(H)2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H)1 immunity.

Antidepressant-like effects of a water-soluble extract from the culture medium of Ganoderma lucidum mycelia in rats.

BACKGROUND: Ganoderma lucidum is a popular medicinal mushroom used for promoting health and longevity in Asian countries. Previously, we reported that a water-soluble extract from a culture medium of Ganoderma lucidum mycelia (MAK) exerts antioxidative and cerebroprotective effects against ischemia-reperfusion injury in vivo. Here, we evaluated the antidepressant and anxiolytic activities of MAK in rats. METHODS: MAK (0.3 or 1 g/kg, p.o.) was administered in the experimental animals 60 min before the forced swimming, open-field, elevated plus-maze, contextual fear-conditioning, and head twitch tests. Additionally, the mechanisms involved in the antidepressant-like action of MAK were investigated by the serotonin precursor 5-hydroxy-L-tryptophan (5-HTP)- or 5-HT2A agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)-induced head twitch responses. RESULTS: Treatment with MAK (1 g/kg) exhibited antidepressant-like effects in the forced swimming test, attenuated freezing behavior in the contextual fear-conditioning test, and decreased the number of head twitches induced by DOI, but not with 5-HTP. No significant response was observed in locomotion or anxiety-like behavior, when the animals were evaluated in the open-field or elevated plus-maze test, respectively. CONCLUSIONS: These data suggest that MAK has antidepressant-like potential, which is most likely due to the antagonism of 5-HT2A receptors, and possesses anxiolytic-like effects toward memory-dependent and/or stress-induced anxiety in rats.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Gene cloning and characterization of two NADH-dependent 3-quinuclidinone reductases from Microbacterium luteolum JCM 9174.

We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(-)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(-)-3-quinuclidinol.

Novel photosensory two-component system (PixA-NixB-NixC) involved in the regulation of positive and negative phototaxis of cyanobacterium Synechocystis sp. PCC 6803.

Two wild-type substrains of a motile cyanobacterium Synechocystis sp. PCC 6803 show positive phototaxis toward a light source (PCC-P) and negative phototaxis away from light (PCC-N). In this study, we found that a novel two-component system of photoresponse is involved in the phototactic regulation. Inactivation of slr1212 (pixA), which encodes a photoreceptor histidine kinase, reverted the positive phototaxis of PCC-P to negative phototaxis, and inactivation of the downstream slr1213 (nixB) and slr1214 (nixC), which encode AraC-like transcription factor-type and PatA-type response regulators, respectively, reverted the negative phototaxis of PCC-N to positive phototaxis. Opposite effects of pixA and nixBC disruption implies an unexpected signal transduction pathway in the switching of positive and negative phototaxis. The blue/green-type cyanobacteriochrome GAF domain of PixA was expressed in Synechocystis and phycocyanobilin-producing Escherichia coli. The holoprotein covalently bound a chromophore phycoviolobilin and showed reversible photoconversion between the violet- (Pv, λ(peak) = 396 nm) and green-absorbing (Pg, λ(peak) = 533 nm) forms, although the protein from E. coli partially bound a precursor phycocyanobilin. These results were discussed with regard to an idea that PixA serves as a violet light receptor for switching of positive and negative phototaxis by transcriptional and functional regulation.

Identification of cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) as a novel alpha1A-adrenoceptor-down-regulating protein and establishment of an alpha1L-adrenoceptor-expressing cell line.

Two distinct alpha(1)-adrenoceptor phenotypes (alpha(1A)- and alpha(1L)-ARs) are known to originate from a single ADRA1A(alpha(1a)) gene by an as-yet-unknown mechanism. We hypothesized that an alpha(1a)-AR-interacting protein could generate the alpha(1L)-AR phenotype and we sought to identify such a protein and to examine its effects on the expression of alpha(1A) and alpha(1L) phenotypes. Cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) was first identified using a yeast two-hybrid approach as an alpha(1a)-AR-interacting protein. Transfection of alpha(1a)-AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing alpha(1A)-ARs having a predominant high affinity site for prazosin, with a low proportion (<10%) of prazosin-low affinity sites (alpha(1L)-AR). Knockdown of endogenous CHO-CRELD1alpha [alpha(1a)-CKD(alpha(1A)-enhanced) cells] enhanced the expression of alpha(1A)-AR, whereas over-expression of CRELD1alpha reduced alpha(1A)-AR expression, yielding alpha(1a)-COE(alpha(1L)-dominant) cells expressing a high proportion (50%) of the alpha(1L)-AR phenotype. The ligand binding and functional agonist and antagonist profiles in alpha(1a)-CKD(alpha(1A)-enhanced) and alpha(1a)-COE(alpha(1L)-dominant) cell lines were entirely in accord with the alpha(1A)-AR and alpha(1L)-AR phenotypes observed in intact tissues. CRELD1alpha down-regulates expression of the alpha(1A)-AR, thereby enhancing the proportion of expression of the alpha(1L)-AR phenotype. The alpha(1L)-AR-expressing alpha(1a)-COE(alpha(1L)-dominant) cell line reflects accurately the phenotype of this AR observed in vivo and will facilitate development of alpha(1L)-AR-targeted drugs.

Assessment of novel muscarinic acetylcholine receptors in rat cerebral cortex by a tissue segment binding method.

Muscarinic acetylcholine receptors (mAChRs) of rat cerebral cortex were evaluated using a tissue segment radioligand binding assay. [(3)H]-Quinuclidinyl benzilate (QNB, a hydrophobic ligand) specifically bound to mAChRs in the cortex segments. The total mAChRs level was approximately 2,000 fmol/mg protein, which was estimated after incubation for 120 min at 37 degrees C or for 8 h at 4 degrees C. These mAChRs were a mixture of high- and low-affinity sites for N-methylscopolamine (NMS) in a 70:30 ratio. In contrast, only a single high-affinity site for NMS was detected following incubation for 30 min at 37 degrees C, whose abundance was about 70% of that of the total mAChRs. Atropine showed a single affinity for mAChRs under all conditions. These indicate that mAChRs are constitutively expressed not only on plasma membrane sites but also at intracellular sites in rat cerebral cortex and that the receptors at both sites have different affinities for NMS. Acetylcholine completely inhibited [(3)H]-QNB binding to both mAChRs without any change in the subcellular distribution, suggesting the possibility that acetylcholine can access, and bind to, both mAChRs in intact tissue. Two different affinity states for acetylcholine were detected only in plasma membrane mAChRs at 37 degrees C. The present study demonstrates a unique subcellular distribution, and distinct pharmacological profiles, of mAChRs in rat cerebral cortex.

Alpha 1-adrenoceptor pharmacome: alpha 1L-adrenoceptor and alpha 1A-adrenoceptor in the lower urinary tract.

Alpha(1)-adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several alpha(1) antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) have been identified, among which the alpha(1A) and alpha(1D)-adrenoceptor subtypes have been regarded as the main targets of alpha(1) antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize alpha(1)-adrenoceptors pharmacologically, (i.e. all classical alpha(1)-adrenoceptor subtypes show high-affinity for the drug). However, we found that alpha(1)-adrenoceptors in the LUT show atypical low-affinity for prazosin. Therefore, the concept alpha(1L)-receptor, which indicates alpha(1)-adrenoceptor(s) showing low-affinity for prazosin has been introduced. A recent study demonstrated that the alpha(1L)-adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the alpha(1L)-adrenoceptor in the LUT is now re-defined as alpha(1A(L))-adrenoceptor. The physiological and pharmacological difference between classical alpha(1A(H),) and alpha(1A(L)) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological alpha(1A)-adrenoceptor blocking pharmacotherapy. Here, we briefly review the alpha(1)-adrenoceptors in the LUT with special reference to phenotype-based (pharmacome) analysis.