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1.
Cell Mol Neurobiol ; 2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32856232

RESUMO

Prothymosin alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca2+-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca2+-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca2+-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.

2.
Cells ; 9(8)2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32824296

RESUMO

Lysophosphatidic acid (LPA) signaling is known to play key roles in the initiation and maintenance of various chronic pain models. Here we examined whether LPA signaling is also involved in diabetes-induced abnormal pain behaviors. The high-fat diet (HFD) showing elevation of blood glucose levels and body weight caused thermal, mechanical hyperalgesia, hypersensitivity to 2000 or 250 Hz electrical-stimulation and hyposensitivity to 5 Hz stimulation to the paw in wild-type (WT) mice. These HFD-induced abnormal pain behaviors and body weight increase, but not elevated glucose levels were abolished in LPA1-/- and LPA3-/- mice. Repeated daily intrathecal (i.t.) treatments with LPA1/3 antagonist AM966 reversed these abnormal pain behaviors. Similar abnormal pain behaviors and their blockade by daily AM966 (i.t.) or twice daily Ki16425, another LPA1/3 antagonist was also observed in db/db mice which show high glucose levels and body weight. Furthermore, streptozotocin-induced similar abnormal pain behaviors, but not elevated glucose levels or body weight loss were abolished in LPA1-/- and LPA3-/- mice. These results suggest that LPA1 and LPA3 play key roles in the development of both type I and type II diabetic neuropathic pain.

3.
Biosens Bioelectron ; 165: 112425, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32729542

RESUMO

Quenchbody (Q-body) is a fluorescent biosensor in which a fluorescent dye is tagged near the antigen binding site of an antibody. The fluorescence of the dye is quenched by the tryptophan residues present in the variable region of the antibody, and is recovered when the antigen binds. Q-bodies have been prepared using recombinant DNA technology by introducing one or more tag sequence(s) at either the N-terminal of the Fab or the single chain variable region fragment of the antibody, and labeling the tag with a fluorescent dye. However, preparation of recombinant antibody fragments is time-consuming and the performance of the Q-body is unpredictable. Here we report an antibody-binding quenching probe made from protein M from Mycoplasma genitalium that can transform the IgG antibody into an immunosensor. By using bacterially expressed and purified protein M and labeling the C-terminal cysteine-containing tag, we prepared a TAMRA-labeled PM Q-probe. When the Q-probe was incubated with Fab or IgG recognizing the bone Gla protein, the fluorescence of the probe was quenched and subsequently recovered by the adding of antigens in a dose-dependent manner. We also succeeded in detecting several small biomarkers with nanomolar sensitivity, including thyroxine extracted from human serum. The clone found to be suitable for the detection of cortisol was confirmed to work as a recombinant Q-body as well, which also worked in 50% human serum. The results suggest that the Q-probe can quickly convert an IgG to a biosensor, which will be useful in rapid diagnosis of small biomarkers.

4.
Biomacromolecules ; 21(8): 3017-3025, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32659086

RESUMO

Current slow-freezing methods are too inefficient for cryopreservation of three-dimensional (3D) tissue constructs. Additionally, conventional vitrification methods use liquid nitrogen, which is inconvenient and increases the chance of cross-contamination. Herein, we have developed polyampholytes with various degrees of hydrophobicity and showed that they could successfully vitrify cell constructs including spheroids and cell monolayers without using liquid nitrogen. The polyampholytes prevented ice crystallization during both cooling and warming, demonstrating their potential to prevent freezing-induced damage. Monolayers and spheroids vitrified in the presence of polyampholytes yielded high viabilities post-thawing with monolayers vitrified with PLL-DMGA exhibiting more than 90% viability. Moreover, spheroids vitrified in the presence of polyampholytes retained their fusibilities, thus revealing the propensity of these polyampholytes to stabilize 3D cell constructs. This study is expected to open new avenues for the development of off-the-shelf tissue engineering constructs that can be prepared and preserved until needed.

5.
Int J Pharm ; 587: 119662, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32682958

RESUMO

Co-amorphous systems comprising low-molecular weight drugs and co-formers constitute an interesting approach to optimize pharmaceutical performance of drugs with low aqueous solubility. Within the different types of co-amorphous systems, the combination of a drug with its own salt may be an attractive formulation option due the absence of any inactive co-formers. The aim of this study was to investigate the possibility of forming a co-amorphous system from naproxen (NAP) and its sodium salt (NAP(Na)). Ball milling of NAP and NAP(Na) at equal molar ratio resulted in the formation of a co-amorphous system whilst NAP and NAP(Na) alone were crystalline following both, ball milling and melt quenching. Infrared spectroscopy and physical stability testing revealed that intermolecular interactions were able to maintain the ball milled NAP-NAP(Na) system amorphous for 2 months at 40 °C. Surprisingly, the dissolution rate of co-amorphous NAP-NAP(Na) was only intermediate between those of crystalline NAP and crystalline NAP(Na). In situ Raman spectroscopic measurements indicated an initial phase separation of the co-amorphous form to NAP and NAP(Na) followed by dissociation of sodium from NAP(Na) and crystallization to NAP. These findings contribute to the design of co-amorphous formulations with the combination of a drug and its own salt.

6.
J Pharmacol Exp Ther ; 375(1): 1-9, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32665319

RESUMO

Treatment of fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly understood. In a series of studies, we have demonstrated that some pharmacological treatments reverse generalized chronic pain but do not affect the lack of morphine analgesia in the intermittent cold stress (ICS)-induced fibromyalgia-like pain model in mice. Here we report that repeated intraperitoneal treatments with mirtazapine, which is presumed to disinhibit 5-hydroxytriptamine (5-HT) release and activate 5-HT1 receptor through mechanisms of blocking presynaptic adrenergic α2 and postsynaptic 5-HT2 and 5-HT3 receptors, completely reversed the chronic pain for more than 4 to 5 days after the cessation of treatments. The repeated mirtazapine treatments also recovered the morphine analgesia after the return of nociceptive threshold to the normal level. The microinjection of small interfering RNA (siRNA) adrenergic α2a receptor (ADRA2A) into the habenula, which showed a selective upregulation of α2 receptor gene expression after ICS, reversed the hyperalgesia but did not recover the morphine analgesia. However, both reversal of hyperalgesia and recovery of morphine analgesia were observed when siRNA ADRA2A was administered intracerebroventricularly. As the habenular is reported to be involved in the emotion/reward-related pain and hypoalgesia, these results suggest that mirtazapine could attenuate pain and/or augment hypoalgesia by blocking the habenular α2 receptor after ICS. The recovery of morphine analgesia in the ICS model, on the other hand, seems to be mediated through a blockade of α2 receptor in unidentified brain regions. SIGNIFICANCE STATEMENT: This study reports possible mechanisms underlying the complete reversal of hyperalgesia and recovery of morphine analgesia by mirtazapine, a unique antidepressant with adrenergic α2 and serotonergic receptor antagonist properties, in a type of intermittently repeated stress (ICS)-induced fibromyalgia-like pain model. Habenula, a brain region which is related to the control of emotional pain, was found to play key roles in the antihyperalgesia, whereas other brain regions appeared to be involved in the recovery of morphine analgesia in the ICS model.

7.
J Chem Inf Model ; 60(7): 3361-3368, 2020 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-32496771

RESUMO

Here, we have constructed neural network-based models that predict atomic partial charges with high accuracy at low computational cost. The models were trained using high-quality data acquired from quantum mechanics calculations using the fragment molecular orbital method. We have succeeded in obtaining highly accurate atomic partial charges for three representative molecular systems of proteins, including one large biomolecule (approx. 2000 atoms). The novelty of our approach is the ability to take into account the electronic polarization in the system, which is a system-dependent phenomenon, being important in the field of drug design. Our high-precision models are useful for the prediction of atomic partial charges and expected to be widely applicable in structure-based drug designs such as structural optimization, high-speed and high-precision docking, and molecular dynamics calculations.

8.
Bioconjug Chem ; 31(5): 1417-1424, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32223219

RESUMO

Targeting less abundant amino acid residues on the protein surface may realize site-selective protein modification of natural proteins. The relative hydrophobicity of tyrosine combined with the π-π stacking tendency of the aromatic rings results in generally low accessibility. In this study, site-selective protein modification was achieved by targeting surface-exposed tyrosine residues without using a genetic encoding system. Tyrosine residues were modified with N-methylated luminol derivative under single-electron transfer (SET) reaction conditions. Horseradish peroxidase (HRP)-catalyzed SET and electrochemically activated SET modified surface-exposed tyrosine residues selectively. N-Methylated luminol derivative modified tyrosine residues more efficiently than 4-arylurazole under tyrosine click conditions using HRP and electrochemistry. Tyrosine residues that are evolutionarily exposed only in the complementarity-determining region (CDR) of an antibody were selectively modified by tyrosine click reactions. CDR-modified antibodies were applied to in vivo imaging and antibody-drug conjugated (ADC).

9.
J Pharmacol Sci ; 143(2): 127-131, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32156464

RESUMO

The inhibition of retinal ischemia-induced damage by post-ischemic prothymosin alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or ß-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.


Assuntos
Isquemia/metabolismo , Isquemia/prevenção & controle , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Retina , Timosina/análogos & derivados , Animais , Hidrólise/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Recombinantes/metabolismo , Timosina/administração & dosagem , Timosina/farmacologia
10.
Biotechnol Bioeng ; 117(5): 1259-1269, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32039472

RESUMO

Ultra Quenchbody (UQ-body) is a biosensor that utilizes the quenching behavior of the fluorescent dye linked to the antibody V region. When the corresponding antigen is bound to the UQ-body, the fluorescence is restored and allows the detection of target molecules easily and sensitively. In this paper, we constructed UQ-bodies to sensitively detect the human epidermal growth factor receptor 2 (HER2) cancer marker in solution or on cancer cells, which was further used to kill the cancer cells. A synthetic Fab fragment of anti-HER2 antibody Fab37 with many Trp residues at hypervariable region was prepared and labeled with fluorescent dyes to obtain the UQ-bodies. The UQ-body could detect HER2 in solution at concentrations as low as 20 pM with an EC50 of 0.3 nM with a fourfold response. Fluorescence imaging of HER2-positive cells was successfully performed without any washing steps. To deliver small interfering RNA (siRNA) to cancer cells, a modified UQ-body with C-terminal 9R sequence was also prepared. HER2-positive cancer cells were effectively killed by polo-like kinase 1 siRNA intracellularly delivered by the UQ-body-9R. The novel approach employing siRNA-empowered UQ-body could detect and image the HER2 antigen easily and sensitively, and effectively kill the HER2-positive cancer cells.

11.
Artigo em Inglês | MEDLINE | ID: mdl-32022534

RESUMO

The increasing demand for high-energy-density batteries stimulated the revival of research interest in Li-metal batteries. The garnet-type ceramic Li7La3Zr2O12 (LLZO) is one of the few solid-state fast-ion conductors that are stable against Li metal. However, the densification of LLZO powders usually requires high sintering temperatures (e.g., 1200 °C), which likely result in Li loss and various side reactions. From an engineering point of view, high-temperature sintering of thin LLZO electrolytes (brittle) at a large scale is difficult. Moreover, the high interfacial resistance between the solid LLZO electrolytes and electrodes is a notorious problem. Here, we report a practical synthesis of a flexible composite Al-doped LLZO (Al-LLZO) sheet electrolyte (75 µm in thickness), which can be mass-produced at room temperature. This ceramic-based flexible sheet electrolyte enables Li-metal batteries to operate at both 60 and 30 °C, demonstrating its potential application for developing practical Li-metal batteries.

12.
Clin Exp Nephrol ; 24(5): 411-419, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31912273

RESUMO

BACKGROUND: Prothymosin alpha (ProTα) is a nuclear protein expressed in virtually all mammalian tissues. Previous studies have shown that ProTα exhibits protective effects against ischemia-induced cell death in various cell types. Recently, the 6-residue peptide P6Q (NEVDQE), the modified form of the active 6-residue core (51-56) in ProTα, has also been shown to have protective effects against retinal ischemia. However, it remains to be elucidated whether P6Q is effective against acute kidney injury (AKI). Therefore, we investigated the renoprotective effect of P6Q on cisplatin-induced AKI. METHODS: Cultured HK-2 cells were treated with cisplatin for 24 h and pretreatment with ProTα or P6Q was carried out 30 min before cisplatin treatment. Cell viability was evaluated using the MTT assay. In an in vivo study, 8-week-old male Wistar rats were divided into control, cisplatin treated, and cisplatin treated with P6Q injection groups. In the last of these, P6Q was injected intravenously before cisplatin treatment. Then, we evaluated the renoprotective effect of P6Q. RESULTS: In the study on cultured cells, pretreatment with ProTα or P6Q prevented cisplatin-induced cell death. In the in vivo study, pretreatment with P6Q significantly attenuated cisplatin-induced increase in serum creatinine and blood urea nitrogen levels, renal tubular cell injury, and apoptosis. Moreover, P6Q attenuated the mitochondrial apoptotic pathway and accelerated Akt phosphorylation after cisplatin-induced renal damage. CONCLUSION: Taken together, our findings indicate that P6Q can attenuate cisplatin-induced AKI and suppress the mitochondrial apoptotic pathway via Akt phosphorylation. These data suggest that P6Q has potential as a preventative drug for cisplatin-induced AKI.

13.
ACS Sens ; 5(1): 24-28, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31922395

RESUMO

Due to its high carrier mobility, graphene is considered a suitable material for use in field-effect transistors. However, its application to immunosensing of small molecules is still elusive. To investigate the potential of graphene field effect transistors (G-FET) as a sensor for small molecules with small or no charge, we applied the open-sandwich immunoassay (OS-IA), which detects low-molecular-weight antigens noncompetitively, to G-FET. Using an antibody variable fragment VL immobilized on graphene and a hyperacidic region of amyloid precursor protein fused to the other variable fragment VH, we successfully detected a small antigen peptide consisting of 7 amino acids (BGP-C7), with a more than 100-fold increase in sensitivity compared with that measured by enzyme-linked OS-IA. Furthermore, we succeeded in detecting BGP-C7 in the presence of human serum with similar sensitivity, suggesting its potential application in clinical diagnostics.

14.
J Pharmacol Exp Ther ; 373(1): 103-112, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31941720

RESUMO

We have developed an experimental fibromyalgia-like mouse model using intermittent cold stress (ICS), where chronic pain is generalized, female predominant, and abolished in type 1 lysophosphatidic acid receptor-knockout (LPA1 -/-) mice but is not reversed by systemic or brain treatment with morphine. We investigated two issues in the present study: (1) whether chronic pain mechanisms and lack of brain morphine analgesia are associated in the ICS model and (2) what mechanisms are involved in the lack of morphine analgesia. ICS-induced hyperalgesia was not affected in µ-opioid receptor-knockout (MOPr -/-) mice, whereas the lack of brain morphine analgesia remained unchanged in LPA1 -/- mice, which completely abolished the hyperalgesia in the ICS model. In contrast, the lack of morphine analgesia was abolished in NR2A-NMDA receptor-knockout (NR2A -/- ) mice and blocked by intracerebroventricular (i.c.v.) injection of (R)-CPP, an NR2A antagonist, or by microinjection of siRNA NR2A into the periaqueductal gray matter region, whereas no change was observed with Ro 04-5595, an NMDA receptor subtype 2B antagonist (i.c.v.). The lack of morphine analgesia was also reversed by concomitant treatment with 1 mg/kg intraperitoneal (i.p.) of dextromethorphan, which possesses NMDA receptor antagonist activity but no analgesic activity. Finally, the hyperalgesia was completely reversed by methadone, which possesses both MOPr agonist and NMDA receptor antagonist activity. Indeed, methadone analgesia was abolished in MOPr -/- mice. These results suggest that chronic pain status and lack of morphine analgesia are independent of each other, and that lack of morphine analgesia is mediated by activation of the NR2A-NMDA receptor system. SIGNIFICANCE STATEMENT: This study reports that a type of intermittently repeated stress causes widespread pain that does not respond to morphine. Because this lack of morphine analgesia is not affected in mice, in which chronic pain is abolished, the mechanisms underlying chronic pain and lack of morphine analgesia are independent of each other. Through speculation that a lack of morphine analgesia may be a secondary event to endogenous opioid analgesic tolerance, the authors demonstrate that an antiopioid N-methyl-D-aspartate receptor system counterbalances the µ-opioid receptor-mediated analgesic mechanisms in the intermittent cold stress model.


Assuntos
Analgésicos Opioides/uso terapêutico , Dor Crônica/tratamento farmacológico , Modelos Animais de Doenças , Fibromialgia/tratamento farmacológico , Morfina/uso terapêutico , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Analgésicos Opioides/farmacologia , Animais , Dor Crônica/metabolismo , Temperatura Baixa/efeitos adversos , Fibromialgia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo
15.
Peptides ; 126: 170265, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31982448

RESUMO

Prothymosin alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.

16.
Biol Pharm Bull ; 43(3): 393-398, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31801924

RESUMO

The aim of this study was to prepare binary supercooled liquid (SCL) by intermolecular interaction and apply this formulation to transdermal drug delivery. Ketoprofen (KET) and ethenzamide (ETH) were selected as binary SCL component. Thermal analysis of physical mixtures of KET and ETH showed decreases in melting points and glass transition below room temperature, thereby indicating formation of KET-ETH SCL. Intermolecular interactions between KET and ETH in the SCL were evaluated from Fourier transform (FT)-IR spectra. KET-ETH SCL maintained SCL state at 25°C with silica gel over 31 d and at 40°C/89% relative humidity (RH) over 7 d. KET SCL and KET-ETH SCL showed similar permeability of KET for hairless mice skin, which was two-fold higher than that of KET aqueous suspension. Our findings suggest that the SCL state could enhance the skin permeation of drugs and the binary SCL formed by intermolecular interaction could also improve the stability of the SCL. The binary SCL system could become a new drug form for transdermal drug delivery.

17.
Biochem Biophys Res Commun ; 522(1): 264-269, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31759625

RESUMO

We previously showed that prothymosin alpha (ProTα) improves cerebral ischemia-induced motor dysfunction. Our recent study also demonstrated that heterozygous ProTα deletion exhibited an enhanced anxiety-like behavior in mice. However, it remains elusive which brain regions or cells are related to these phenotypes. Here we generated conditional Gγ7-specific ProTα knockout mice using G protein γ7 subunit gene (Gng7)-cre promoter to see the brain robustness roles of ProTα in the striatum and hippocampus. The younger conditional ProTα (Gng7) knockout mice at the age of 10 weeks showed no significant phenotypes in motor dysfunction in the Rotarod test and locomotor activity in the open-field test, whereas significant motor dysfunction was obtained by 15 min transient middle cerebral artery occlusion (tMCAO)-induced cerebral ischemia. The aged conditional ProTα (Gng7) knockout mice at the age of 20 weeks showed hypolocomotor activity with less center time in the open-field test and impaired motor coordination in the Rotarod test without ischemia. Thus, this study suggests that ProTα has important roles in the maintenance of motor coordination and anxiety-like behavior.

18.
Pain Manag ; 10(1): 43-53, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31852400

RESUMO

Since the first discovery that the bioactive lipid, lysophosphatidic acid (LPA) and LPA1 receptor signaling play a role in the initiation of neuropathic pain (NeuP), accumulated reports have supported the original findings and extended the study toward possible therapeutic applications. The present review describes beneficial roles of LPA receptor signaling in a variety of chronic pain, such as peripheral NeuP induced by nerve injury, chemotherapy and diabetes, central NeuP induced by cerebral ischemia with hemorrhage and spinal cord injury, and fibromyalgia-like wide spread pain induced by repeated cold, psychological and muscular acidic stress. Emerging mechanistic findings are the feed-forward amplification of LPA production through LPA1, LPA3 and microglia and the evidence for maintenance of chronic pain by LPA receptor signaling.

19.
Sci Rep ; 9(1): 18189, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796769

RESUMO

It is known that digital counting of fluorescent signals generated in many small compartments can significantly improve the detection sensitivity of the enzyme-linked immunosorbent assay (ELISA). However, the reported digital ELISA systems need extensive washing steps to remove background signal, which hampers their performance. To tackle this problem, we developed a vesicle (Protocell) array wherein binding of an external protein analyte is coupled to signal amplification and intra-vesicular fluorescence readout. We chose ß-glucuronidase (GUS) as a reporter enzyme as its function requires assembly of four subunits through dimerization of a pair of dimers that can be inhibited by a set of interface mutations. Using a thermostabilized GUS mutant IV-5, we screened out an interface mutant (M516K, F517W) to create IV5m - a mutant with high thermostability and activity conditional on induced dimerization. After tethering a short N-terminal tag and transmembrane (TM) sequences, the fusion protein was expressed by cell-free protein synthesis inside protocells. When a corresponding tag-specific antibody was applied outside of the protocells, a clear increase in GUS activity was observed inside vesicles by adding fluorescent substrate, probably due to spontaneous integration of the tagged TM protein into the vesicles and dimerization by the antibody bound to the displayed tag. Furthermore, using flow cytometry, quantitative digital read out was obtained by counting fluorescent protocells exposed to varying concentrations of external antibodies that included Trastuzumab. Additionally, through use of an anti-caffeine VHH-SpyCatcher fusion protein, caffeine could be detected using SpyTag-fused TM-IV5m protein expressed in protocells, suggesting utility of this platform for detection of diverse antigen types.

20.
Yakugaku Zasshi ; 139(11): 1403-1415, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31685737

RESUMO

For my Ph.D. research topic, I isolated endogenous morphine-like analgesic dipeptide, kyotorphin, which mediates Met-enkephalin release, and discovered kyotorphin synthetase, a putative receptor and antagonist. Furthermore, I succeeded in purifying µ-opioid receptor and functional reconstitution with purified G proteins. After receiving my full professor position at Nagasaki University in 1996, I worked on two topics of research, molecular mechanisms of chronic pain through lysophosphatidic acid (LPA) and identification and characterization of neuroprotective protein, prothymosin α. In a series of studies, we have shown that LPA signaling defines the molecular mechanisms of neuropathic pain and fibromyalgia in terms of development and maintenance. Above all, the discovery of feed-forward system in LPA production and pain memory may contribute to better understanding of chronic pain and future analgesic drug discovery. Regarding prothymosin α, we first discovered it as neuronal necrosis-inhibitory molecule through two independent mechanisms, such as toll-like receptor and F0/F1 ATPase, both which protect neurons through indirect mechanisms. Prothymosin α is released by non-classical and non-vesicular mechanisms on various stresses, such as ischemia, starvation, and heat-shock. Thus it may be called a new type of neuroprotective damage-associated molecular patterns (DAMPs)/Alarmins. Heterozygotic mice showed a defect in memory-learning and neurogenesis as well as anxiogenic behaviors. Small peptide, P6Q derived from prothymosin α retains neuroprotective actions, which include blockade of cerebral hemorrhage caused by late treatment with tissue plasminogen activator in the stroke model in mice.


Assuntos
Dor Crônica/etiologia , Dor Crônica/genética , Fármacos Neuroprotetores , Precursores de Proteínas , Receptores de Ácidos Lisofosfatídicos/fisiologia , Transdução de Sinais/fisiologia , Timosina/análogos & derivados , Animais , Endorfinas , Humanos , Camundongos , Precursores de Proteínas/metabolismo , ATPases Translocadoras de Prótons , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores Opioides , Acidente Vascular Cerebral , Timosina/metabolismo , Receptores Toll-Like
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