A New Method for Albuminuria Measurement Using a Specific Reaction between Albumin and the Luciferin of the Firefly Squid Watasenia scintillans.

This study demonstrates that the luciferin of the firefly squid Watasenia scintillans, which generally reacts with Watasenia luciferase, reacted with human albumin to emit light in proportion to the albumin concentration. The luminescence showed a peak wavelength at 540 nm and was eliminated by heat or protease treatment. We used urine samples collected from patients with diabetes to quantify urinary albumin concentration, which is essential for the early diagnosis of diabetic nephropathy. Consequently, we were able to measure urinary albumin concentrations by precipitating urinary proteins with acetone before the reaction with luciferin. A correlation was found with the result of the immunoturbidimetric method; however, the Watasenia luciferin method tended to produce lower albumin concentrations. This may be because the Watasenia luciferin reacts with only intact albumin. Therefore, the quantification method using Watasenia luciferin is a new principle of urinary albumin measurement that differs from already established methods such as immunoturbidimetry and high-performance liquid chromatography.

Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells.

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.

Mice lacking BCAS1, a novel myelin-associated protein, display hypomyelination, schizophrenia-like abnormal behaviors, and upregulation of inflammatory genes in the brain.

The abnormal expression and function of myelin-related proteins contribute to nervous system dysfunction associated with neuropsychiatric disorders; however, the underlying mechanism of this remains unclear. We found here that breast carcinoma amplified sequence 1 (BCAS1), a basic protein abundant in the brain, was expressed specifically in oligodendrocytes and Schwann cells, and that its expression level was decreased by demyelination. This suggests that BCAS1 is a novel myelin-associated protein. BCAS1 knockout mice displayed schizophrenia-like behavioral abnormalities and a tendency toward reduced anxiety-like behaviors. Moreover, we found that the loss of BCAS1 specifically induced hypomyelination and the expression of inflammation-related genes in the brain. These observations provide a novel insight into the functional link between oligodendrocytes and inflammation and/or abnormal behaviors.

Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain.

BACKGROUND: Bioluminescence imaging (BLI) is a powerful technique for monitoring the temporal and spatial dynamics of gene expression in the mouse brain. However, the black fur, skin pigmentation and hair regrowth after depilation of mouse interfere with BLI during developmental and daily examination. The aim of this study was to extend the application of Arc-Luc transgenic (Tg) mice to the BLI of neuronal activity in the mouse brain by introducing the hairless (HL) gene and to examine Arc-Luc expression at various developmental stages without interference from black fur, skin pigmentation, and hair regrowth. RESULTS: The Arc-Luc Tg HL mice were established by crossing the Tg C57BL/6 mouse strain with the HL mouse strain. Under physiological and pathological conditions, BLI was performed to detect the signal intensity changes at various developmental stages and at an interval of <7 days. The established Arc-Luc Tg HL mice exhibited clear and stable photon signals from the brain without interference during development. After surgical monocular deprivation during visual-critical period, large signal intensity changes in bioluminescence were observed in the mouse visual cortex. Exposure of mice to a novel object changed the photon distribution in the caudal and rostral cerebral areas. The temporal pattern of kainic-acid-induced Arc-Luc expression showed biphasic changes in signal intensity over 24 h. CONCLUSIONS: This study showed the advantages of using the mutant HL gene in BLI of Arc expression in the mouse brain at various developmental stages. Thus, the use of the Arc-Luc Tg HL mice enabled the tracking of neuronal-activity-dependent processes over a wide range from a focal area to the entire brain area with various time windows.

Novel role of serine racemase in anti-apoptosis and metabolism.

BACKGROUND: Serine racemase (SR) catalyzes the production of d-serine, a co-agonist of the N-methyl-d-aspartate receptor (NMDAR). A previous report shows the contribution of SR in the NMDAR-mediated neuronal cell death process. METHODS AND RESULTS: To analyze the intrinsic role of SR in the cell death process, we established the epithelial human embryonic kidney 293T (HEK293T) cell lines expressing wild-type SR (SR-WT), catalytically inactive mutant SR (SR-K56G), and catalytically hyperactive mutant SR (SR-Q155D). To these cell lines, staurosporine (STS), which induces apoptosis, was introduced. The cells expressing SR-WT and SR-Q155D showed resistance to STS-induced apoptosis, compared with nontransfected HEK293T cells and cells expressing SR-K56G. The SR-WT cells also showed a significant higher viability than the SR-QD cells. Furthermore, we detected elevated phosphorylation levels of Bcl-2 at serine-70 and Akt at serine-473 and threonine-308, which are related to cell survival, in the cells expressing SR-WT and SR-Q155D. From the results of metabolite analysis, we found elevated levels of acetyl CoA and ATP in cells expressing SR-WT. CONCLUSION: Because SR has two enzymatic activities, namely, racemization and α, ß-elimination, and SR-Q155D shows enhanced racemization and reduced α, ß-elimination activities, we concluded that the racemization reaction catalyzed by SR may have a more protective role against apoptosis than the α, ß-elimination reaction. Moreover, both of these activities are important for maximal survival and elevated levels of acetyl CoA and ATP. GENERAL SIGNIFICANCE: Our findings reveal the NMDAR-independent roles of SR in metabolism and cell survival.

A novel serine racemase inhibitor suppresses neuronal over-activation in vivo.

Serine racemase (SRR) is an enzyme that produces d-serine from l-serine. d-Serine acts as an endogenous coagonist of NMDA-type glutamate receptors (NMDARs), which regulate many physiological functions. Over-activation of NMDARs induces excitotoxicity, which is observed in many neurodegenerative disorders and epilepsy states. In our previous works on the generation of SRR gene knockout (Srr-KO) mice and its protective effects against NMDA- and Aß peptide-induced neurodegeneration, we hypothesized that the regulation of NMDARs' over-activation by inhibition of SRR activity is one such therapeutic strategy to combat these disease states. In the previous study, we performed in silico screening to identify four compounds with inhibitory activities against recombinant SRR. Here, we synthesized 21 derivatives of candidate 1, one of four hit compounds, and performed screening by in vitro evaluations. The derivative 13J showed a significantly lower IC50 value in vitro, and suppressed neuronal over-activation in vivo.

Identification of a Novel Protein Kinase A Inhibitor by Bioluminescence-Based Screening.

We screened inhibitors in the adenylyl cyclase/protein kinase A/cAMP response element binding protein pathway (AC/PKA/CREB pathway) from a 2400 chemical library by a cell-based assay method using bioluminescence probes. We found a compound that inhibited forskolin-induced cAMP response element (CRE)-dependent transcription, the interaction between the kinase-inducible domain (KID) and the interacting domain (KIX), and endogenous CREB phosphorylation. Furthermore, this compound suppressed the activity of the PKA catalytic subunit dose-dependently. On the other hand, this compound did not inhibit forskolin-induced cAMP up-regulation. Taken together, we conclude that we have identified a new PKA inhibitor that binds to the catalytic subunit directly. We also succeeded in shortening the screening protocol by excluding a screening step which was used in a previous method.

In silico and pharmacological screenings identify novel serine racemase inhibitors.

D-Serine is a coagonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptor and its biosynthesis is catalyzed by serine racemase (SR). The overactivation of the NMDA receptor has been implicated in the development of neurodegenerative diseases, strokes, and epileptic seizures, thus, the inhibitors of SR have potential against these pathological states. Here, we have developed novel inhibitors of SR by in silico screening and in vitro enzyme assay. The newly developed inhibitors have lower IC50 value comparing with that of malonate, one of the standard SR inhibitor. The structural features of novel inhibitors suggest the importance of central amide structure having a phenoxy substituent in their structure for the SR inhibitory activity. The present findings suggest the importance and rational development of new drugs for diseases of NMDAR overactivation.

Discovery of novel adenylyl cyclase inhibitor by cell-based screening.

We screened 2400 compounds to find novel inhibitors of the adenylyl cyclase (AC)-protein kinase A (PKA)-cAMP response-element-binding protein (CREB) signaling pathway (AC/PKA/CREB pathway). Using a multistep cell-based screening system employing split luciferase technique, we narrowed down the candidates effectively from 2400 chemical compounds and identified a novel AC inhibitor (compound 1). Since dysregulation of the AC/PKA/CREB pathway is known to cause diseases not only in the nervous system but also in other organs, compound 1 is expected to be developed as a medicine for these diseases.

Regulation of gene expression in retrovirus vectors by X-ray and proton beam radiation with artificially constructed promoters.

BACKGROUND: We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated. METHODS: Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8. RESULTS: Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin. CONCLUSIONS: Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.

Measuring CREB activation using bioluminescent probes that detect KID-KIX interaction in living cells.

The cyclic adenosine monophosphate response element-binding protein (CREB) is a transcription factor that contributes to memory formation. The transcriptional activity of CREB is induced by its phosphorylation at Ser-133 and subsequent interaction with the CREB-binding protein (CBP)/p300. We designed and optimized firefly split luciferase probe proteins that detect the interaction of the kinase-inducible domain (KID) of CREB and the KIX domain of CBP/p300. The increase in the light intensity of the probe proteins results from the phosphorylation of the responsible serine corresponding to Ser-133 of CREB. Because these proteins have a high signal-to-noise ratio and are nontoxic, it has become possible for the first time to carry out long-term measurement of KID-KIX interaction in living cells. Furthermore, we examined the usefulness of the probe proteins for future high-throughput cell-based drug screening and found several herbal extracts that activated CREB.

Control of actin polymerization via reactive oxygen species generation using light or radiation.

Actin is one of the most prevalent proteins in cells, and its amino acid sequence is remarkably conserved from protozoa to humans. The polymerization-depolymerization cycle of actin immediately below the plasma membrane regulates cell function, motility, and morphology. It is known that actin and other actin-binding proteins are targets for reactive oxygen species (ROS), indicating that ROS affects cells through actin reorganization. Several researchers have attempted to control actin polymerization from outside the cell to mimic or inhibit actin reorganization. To modify the polymerization state of actin, ultraviolet, visible, and near-infrared light, ionizing radiation, and chromophore-assisted light inactivation have all been reported to induce ROS. Additionally, a combination of the fluorescent protein KillerRed and the luminescent protein luciferase can generate ROS on actin fibers and promote actin polymerization. These techniques are very useful tools for analyzing the relationship between ROS and cell function, movement, and morphology, and are also expected to be used in therapeutics. In this mini review, we offer an overview of the advancements in this field, with a particular focus on how to control intracellular actin polymerization using such optical approaches, and discuss future challenges.

Real-time monitoring of actin polymerization in living cells using split luciferase.

Real-time monitoring of actin polymerization in living cells is beneficial for characterizing cellular activities such as migration, proliferation, and death. We developed new bioluminescence-based probe proteins that enable the monitoring of actin polymerization in living cells. Unlike other ordinary split luciferase probes, our probes were incorporated in endogenous actin filament that enabled it to measure the actin polymerization quantitatively. The probe proteins exhibited a dose-responsive decrease in photon emission intensity in response to the filamentous (F)-actin-disrupting agent latrunculin A. This technique has a high sensitivity with a high signal-to-noise ratio and is nontoxic compared with other methods of monitoring actin polymerization in living cells. Using this technique, we succeeded in monitoring the F-actin level in living cells during apoptosis progression induced by UV irradiation continuously for 12 h. F-actin was transiently upregulated after UV irradiation. Since UV-induced cell death was enhanced by treatment with latrunculin A during the period which F-actin is increased, transient upregulation of F-actin after UV is likely a protective reaction against UV-induced cell death. Our novel technique is an effective tool for investigating actin polymerization in living cells.

Manipulation of Actin Cytoskeleton by Intracellular-Targeted ROS Generation.

A method to generate small amount of reactive oxygen species (ROSs) at intracellular targeted region has great potential to manipulate the function of particular proteins. The present protocol introduces a fusion protein that consisted of firefly luciferase (FLuc), photosensitizer protein KillerRed and F-actin-targeting peptide Lifeact (Lifeact-KillerFirefly) to generate ROSs in the vicinity of F-actin and found that morphological change in F-actin structure was induced by the fusion protein after luciferin treatment. This manipulating and imaging method is of use to analyze the role of the locally generated ROSs on the function of intracellular proteins.

Cloning, bacterial expression, and unique structure of adenosylhomocysteine hydrolase-like protein 1, or inositol 1,4,5-triphosphate receptor-binding protein from mouse kidney.

Adenosylhomocysteine hydrolase (SAHase)-like protein 1 (SAH-L), also called inositol 1,4,5-triphosphate receptor-binding protein (IRBIT) is a novel protein involved in fish embryo development and calcium release in mammalian cells through protein-protein interactions. To better understand its reaction mechanism, purified protein is indispensable. Here we describe a simple purification procedure and the unique properties of SAH-L. The cDNA was isolated from mouse kidney by RT-PCR and inserted into various pETtrade mark vectors. Escherichia coli harboring a plasmid coding for SAH-L with a C-terminal His-tag could solely produce a soluble protein. SAH-L purified through a Ni(2+) column gave M(r)s of 59,000 and 190,000 by SDS-PAGE and gel filtration, respectively, which is suggestive of a trimer, but chemical cross-linking experiments demonstrated a dimer. The incompatible M(r) values implicate an irregular structure of SAH-L. In fact, SAH-L was partially purified in a form lacking the 31 N-terminal residues, and was found to be extremely susceptible to proteases in the region around residue 70. The N-terminal polypeptide (residues 1-98) was also expressed as a soluble form and was trypsin-sensitive. Circular dichroism revealed a low alpha-helix content but not a randomly extended structure. Interestingly, SAH-L contained tightly bound NAD(+) despite showing no SAHase activity. The characterized properties of SAH-L and its N-terminal fragment present the notion that the structure of the protease-sensitive N-terminal region is relatively loose and flexible rather than compact, and which protrudes from the major SAHase-like domain. This structure is supposed to be favorable to interact with the IP(3) receptor.

Enhancement of artificial promoter activity by ultrasound-induced oxidative stress.

We previously developed artificial promoters that were activated in response to X-ray irradiation. Sonication with 1.0MHz ultrasound that causes intracellular oxidative stress was found to activate some of these promoters though to lesser degrees. The most sensitive one among these promoters showed intensity- and duration-dependent activations by sonication. In addition, its activation by sonication was attenuated when N-acetyl cysteine was present, suggesting the involvement of intracellular oxidative stress in the activation mechanism. Improved promoters for sensitivity to X-ray irradiation were also found more sensitive to sonication. The most improved one showed 6.0 fold enhancement after sonication with 1.0MHz ultrasound at 1.0W/cm2 for 60s. This enhancement was also attenuated with the presence of N-acetyl cysteine. When stably transfected HeLa cells with the most sensitive promoter were transplanted on to mice and sonicated, luciferase activity by the promoter increased to 1.35 fold in average though it was not statistically significant compared to control. Although gene regulation in vivo by sonication was not clear, this is the first report on artificially constructed promoters responsive to ultrasound.

A new bioluminescence-based tool for modulating target proteins in live cells.

We have developed a new genetically encoded tool designed to generate reactive oxygen species (ROS) at target proteins in cultured cells; it is designed using firefly luciferase and photosensitiser protein KillerRed. Targeting this fusion protein, KillerFirefly, to F-actin in live cells and treatment with luciferin induced a characteristic structure, previously reported as a cofilin-actin rod, which is seen in patients with Alzheimer's disease. This structural change is considered to be elicited by the consistent generation of very low-level ROS by KillerFirefly in the vicinity of F-actin. Moreover, our results suggest the presence of an actin-regulating system, controlled by very low levels of endogenously generated ROS.

Bioluminescence imaging of Arc expression in mouse brain under acute and chronic exposure to pesticides.

Exposure to pesticides can induce neurobehavioral effects in rodents, as well as in other mammals, including humans. However, the effects of the toxicity of pesticides on the central nervous system (CNS) remain largely unclear. The expression of the activity-regulated cytoskeleton-associated protein gene (Arc) is induced in a neuronal-activity-dependent manner and is implicated in synaptic and experience-dependent plasticity. We previously developed Arc-promoter-driven luciferase transgenic (Tg) mouse strains to monitor the neuronal-activity-dependent gene expression under physiological and pathological conditions in vivo. In this study, we examined the effect of acute administration of four different pesticides (deltamethrin, glufosinate, methylcarbaryl, and imidacloprid) on neuronal activity using Arc-Luc Tg mice. The change in the bioluminescence signal in mouse brain upon treatment with deltamethrin and glufosinate occurred more slowly than that of kainic acid, a potent neuroexcitatory amino acid agonist. These two pesticides also caused convulsive responses in adult Arc-Luc Tg mice. In the case of glufosinate, we detected the long-term upregulation of bioluminescence signal intensity of Arc-Luc over 24 h after the treatment. Furthermore, we observed greater changes of bioluminescence signal in adults than in juveniles, and a lower incidence of convulsions at the juvenile stage. In contrast to the acute treatment, we detected a decrease of bioluminescence signal after low-dose chronic treatment with glufosinate, without neuronal overexcitation. From these results, we suggest that Arc-Luc Tg mice are useful for assessing the acute and chronic effects of pesticides on the CNS.

Cloning and characterization of a novel synaptosome-enriched mRNA that encodes 31 kDa protein.

Although a subpopulation of mRNAs has been identified as translocated to the dendrites or the synaptic regions of neurons, the translocational mechanism has not been elucidated. To find mRNAs enriched in synapses, we compared the synaptosomal mRNAs with those from whole forebrain using differential display (DD). We cloned one of these mRNAs, which encoded a novel 31 kDa protein (PMES-2). PMES-2 mRNA was specifically transcribed in the brain and was present in the dendrites of the hippocampal neurons. PMES-2 protein was partly localized in the postsynaptic density. Although this protein is very similar to human NABC1 protein, its function is still unknown.

In vivo imaging of CREB phosphorylation in awake-mouse brain.

The cyclic adenosine monophosphate response element binding protein (CREB) is a phosphorylation-dependent transcription factor that plays important roles in memory consolidation and several neuropsychological disorders. Although analyzing the spatiotemporal pattern of CREB phosphorylation is required for elucidating the mechanism of memory consolidation, imaging of phosphorylation of a particular protein in the brain of live animals is impossible at present. Here, we developed a method for visualizing the CREB phosphorylation in the cerebral cortex of an awake mouse using a split luciferase technique. Using this technique, we demonstrated the correlation between the change in CREB phosphorylation at a particular region in the brain and behavioral consequences induced by the administration of reserpine, a psychotropic agent.