Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

In vitro validation of chromogenic substrate assay for evaluation of surrogate FVIII-activity of emicizumab.

[Introduction] Emicizumab, a bispecific antibody mimicking activated factor VIII (FVIII), is increasingly used in prophylaxis against bleeding in hemophilia A. Human factor-based chromogenic substrate assay (hCSA) shows concentration-dependency between emicizumab and reported FVIII activity. However, the assay measurement settings have not been optimized for emicizumab, and the reported FVIII activity cannot be directly referred as surrogate FVIII activity. [Materials and Methods] For in vitro validation of hCSA-reported surrogate FVIII activity, we compared the equation curves for emicizumab concentration with surrogate FVIII activity using spiked plasma in the thrombin generation assay (TGA), hCSA, and clot waveform analysis (CWA). Then, we generated conversion equations for hCSA-reported surrogate FVIII value to that of TGA. We also assessed the additive effect of rFVIII onto 340 nM (i.e., 50 µg/mL) emicizumab using the same assays. [Results] With 1:20 diluted plasma, halving hCSA-reported surrogate FVIII activity can be approximated to that in TGA triggered by the extrinsic pathway reagent (27.3 IU/dL vs. 13.9 IU/dL) under therapeutic emicizumab concentration. Both in TGA and hCSA, the additive effect of added FVIII on therapeutic emicizumab concentration (340 nM) was maintained at low levels of FVIII but gradually decreased at higher levels. [Conclusions] Surrogate FVIII activity can be estimated simply by halving hCSA-reported FVIII value, and the additive effect of FVIII on emicizumab diminishes at high concentrations. Based on our in vitro study, a clinical study is currently being conducted to compare individual variation of surrogate FVIII activity in hCSA and TGA.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions.

We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope.

Effects of salicylate derivatives on localization of p.H723R allele product of SLC26A4.

OBJECTIVE: Pendrin is a transmembrane protein encoded by the SLC26A4 gene that functions in maintaining ion concentrations in the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Variants in the SLC26A4 gene are responsible for sensorineural hearing loss. Although pendrin localizes to the plasma membrane, we previously identified that 8 missense allele products of SLC26A4 were retained in the intracellular region and lost their anion exchange function. We also found that 10 mM salicylate induced the translocation of 4 out of 8 allele products from the intracellular region to the plasma membrane and restored their anion exchanger activity. However, since 10 mM salicylate exhibits cytotoxicity, the use of chemical compounds with less cell toxicity is needed. In the present study, therefore, salicylate derivatives were used as the chemical compounds and their effects on the p.H723R allele products of SLC26A4 were investigated. METHODS: HEK293 cells were transfected with the cDNA of p.H723R. Cell proliferation, viability and toxicity assays were performed to investigate the response and health of cells in culture after treatment with four types of salicylate derivatives, i.e., 2-hydroxybenzyl alcohol, 2,3-dihydroxybenzoic acid, 2'-hydroxyacetophenone and methyl salicylate. The effects of these salicylate derivatives on the localization of the p.H723R were investigated by immunofluorescence microscopy. RESULTS: The application of 10 mM salicylate showed an increase in cell toxicity and decrease in cell viability, leading to a significant decrease in cell proliferation. In contrast, the application of 1 mM salicylate derivatives did not show any significant increase in cell toxicity and decrease in cell viability, corresponding to a logarithmic increase in cell concentration with an increase in culture time. Immunofluorescence experiments showed that the p.H723R retained in the endoplasmic reticulum (ER). Among the salicylate derivatives applied, 2-hydroxybenzyl alcohol induced the translocation of p.H723R from the ER to the plasma membrane 3 h after its application. CONCLUSION: The results obtained showed that 2-hydroxybenzyl alcohol restored the localization of the p.H723R allele products of SLC26A4 from the ER to the plasma membrane at a concentration of 1 mM by 3 h after its administration with less cytotoxicity than 10 mM salicylate.

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells.

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS-induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild-type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin-binding protein F1-disrupted mutant SAM1-infected cells. In Bcl-2-overexpressing HeLa cells (HBD98-2-4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98-2-4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS-infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.

Two Secreted Proteoglycans, Activators of Urothelial Cell-Cell Adhesion, Negatively Contribute to Bladder Cancer Initiation and Progression.

Osteomodulin (OMD) and proline/arginine-rich end leucine repeat protein (PRELP) are secreted extracellular matrix proteins belonging to the small leucine-rich proteoglycans family. We found that OMD and PRELP were specifically expressed in umbrella cells in bladder epithelia, and their expression levels were dramatically downregulated in all bladder cancers from very early stages and various epithelial cancers. Our in vitro studies including gene expression profiling using bladder cancer cell lines revealed that OMD or PRELP application suppressed the cancer progression by inhibiting TGF-ß and EGF pathways, which reversed epithelial-mesenchymal transition (EMT), activated cell-cell adhesion, and inhibited various oncogenic pathways. Furthermore, the overexpression of OMD in bladder cancer cells strongly inhibited the anchorage-independent growth and tumorigenicity in mouse xenograft studies. On the other hand, we found that in the bladder epithelia, the knockout mice of OMD and/or PRELP gene caused partial EMT and a loss of tight junctions of the umbrella cells and resulted in formation of a bladder carcinoma in situ-like structure by spontaneous breakdowns of the umbrella cell layer. Furthermore, the ontological analysis of the expression profiling of an OMD knockout mouse bladder demonstrated very high similarity with those obtained from human bladder cancers. Our data indicate that OMD and PRELP are endogenous inhibitors of cancer initiation and progression by controlling EMT. OMD and/or PRELP may have potential for the treatment of bladder cancer.

Highly sensitive biomolecular interaction detection method using optical bound/free separation with grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS).

Grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS) with optical bound/free (B/F) separation technique was developed by employing a highly directional fluorescence with polarization of surface plasmon-coupled emission (SPCE) to realize highly sensitive immunoassay regardless of the ligand affinity. A highly sensitive immunoassay system with GC-SPFS was constructed using a plastic sensor chip reproducibly fabricated in-house by nanoimprinting and applied to the quantitative detection of an anti-lysozyme single-domain antibody (sdAb), to compare conventional washing B/F separation with optical B/F separation. Differences in the affinity of the anti-lysozyme sdAb, induced by artificial mutation of only one amino acid residue in the variable domain were attributed to higher sensitivity than that of the conventional Biacore surface plasmon resonance (SPR) system. The detection limit (LOD; means of six replicates of the zero standard plus three standard deviations) of the GC-SPFS immunoassay with optical B/F separation, was estimated to be 1.2 ng/ml with the low-affinity ligand (mutant sdAb Y52A: KD level was of the order of 10-7 ~ 10-6 M) and was clearly improved as compared to that (LOD: 9.4 ng/ml) obtained with the conventional washing B/F separation. These results indicate that GC-SPFS with the optical B/F separation technique offers opportunities to re-evaluate low-affinity biomaterials that are neither fully utilized nor widespread, and could facilitate the creation of novel and innovative methods in drug and diagnostic development.

Improved Brain Expression of Anti-Amyloid ß scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer.

BACKGROUND: The ever-increasing number of people living with Alzheimer's disease urges to develop more effective therapies. Despite considerable success, anti-Alzheimer immunotherapy still faces the challenge of intracerebral and intracellular delivery. This work introduces in situ production of anti-amyloid beta (Aß) antibody after intracerebral injection of PEG-PAsp(DET)/mRNA polyplexes as a novel immunotherapy approach and a safer alternative compared to high systemic antibodies doses or administration of adenovirus encoding anti- Aß antibodies. METHODS: We used mRNA encoding three different Aß-specific scFV with a secretion signal for passive immunotherapy. scFv contained a 6xHis-tag for immuno-detection. The secretion signal from IL2 (IL2ss) was added to allow extracellular engagement of senile plaques. Aß affinity of scFv was measured by surface plasmon resonance. To allow intracellular delivery, scFv were administered as polyplexes formed with our smart copolymer polyethylene glycol-poly[N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PEG-PAsp (DET)]. We evaluated scFv expression in cellulo by Western blot and ELISA, their ability to disaggregate amyloid aggregates by thioflavine T assay. Moreover, in vivo expression and therapeutic activity were evaluated in a murine amyloidosis model, by anti-6xHis-tag ELISA and anti- Aß ELISA, respectively. RESULTS: The selected anti-amyloid beta scFv showed affinity towards Aß and disaggregated Aß fibers in vitro. Whereas both DNA and mRNA transfection led to scFV expression in cancer cells, only mRNA led to detectable scFv expression in primary neurons. In addition, the use of IL2ss increased by 3.4-fold scFv secretion by primary neurons over mRNA polyplexes devoid of secretion signal. In vivo, a 3 to 11- fold of intracranial scFv levels was measured for mRNA compared to DNA polyplexes and higher in vivo scFv levels were obtained with mRNA containing IL2ss over non-secreted mRNA. Intracranial injection of anti-Aß mRNA polyplexes with IL2ss resulted in 40 % Aß decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aß decrease was detected in a more challenging transgenic model of Alzheimer's disease. CONCLUSION: Our results introduce a concerted approach not only for Alzheimer's disease treatment but also for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial delivery of anti-Aß scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aß in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer, IL2ss) were integrated into a single formulation.

Review: Why is arginine effective in suppressing aggregation?

Arginine is finding a wide range of applications in production of proteins. Arginine has been used for many years to assist protein refolding. This effect was ascribed to aggregation suppression by arginine of folding intermediates during protein refolding. Recently, we have observed that arginine facilitates elution of antibodies during Protein-A chromatography and solubilizes insoluble proteins from inclusion bodies, which both can be ascribed to weakening of protein-protein interactions. In order to gain understanding on why arginine is effective in reducing protein-protein interactions and suppressing aggregation, the effects of arginine on stability and solubility of pure proteins have been examined, which showed that arginine is not a protein-stabilizer, but is an aggregation suppressor. However, there is no explanation proposed so far on why arginine suppresses aggregation of proteins. This review addresses such question and then attempts to show differences between arginine and strong denaturants, which are also known as an aggregation suppressor.

Characterization of arginine as a solvent additive: a halophilic enzyme as a model protein.

Arginine suppresses the aggregation of proteins. However, little is known about its mechanism. Here we have used HsNDK (Halobacterium salinarum nucleoside diphosphate kinase) to examine the solvent property of arginine. After exposure to 2 M arginine, HsNDK was diluted to a low salt buffer, resulting in fully active protein. Since unfolded HsNDK cannot refold in such low salt buffer, the observed activity indicates that HsNDK was in the native state in 2 M arginine. Enzyme activity was also examined directly in the presence of arginine, showing that it was active in the presence of 1 M arginine and, to less extent, 2 M arginine. Arginine, however, could not support refolding of heat-denatured HsNDK. HsNDK was stable at 40 degrees C for 19 h incubation in the presence of 1M arginine.

T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

BACKGROUND: The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas. MATERIALS AND METHODS: We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity. RESULTS: The SEA/FUscFv fusion protein retained the reactivity with MK-1-expressing tumor cells, introduced a specific cytotoxicity of lymphokine-activated killer T-cells to the tumor cells, and consequently suppressed the tumor growth in a SCID mouse xenograft model. CONCLUSION: This genetically engineered SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

Salicylate restores transport function and anion exchanger activity of missense pendrin mutations.

The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the homeostasis of the ion concentration of the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. Therefore, in this study, we focused on the function of ten missense pendrin mutations (p.P123S (Pendred syndrome), p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syndrome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA), p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA) and p.H723R (Pendred syndrome/NSEVA)) reported in Japanese patients, and analyzed their cellular localization and anion exchanger activity using HEK293 cells transfected with each mutant gene. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that p.K369E and p.C565Y, as well as wild-type pendrin, were transported to the plasma membrane, while 8 other mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restores normal plasma membrane localization of 8 pendrin mutants retained in the cytoplasm to the plasma membrane. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants (p.P123S, p.M147V, p.S657Y and p.H723R) from the cytoplasm to the plasma membrane and restored the anion exchanger activity. These findings suggest that salicylate might contribute to development of a new method of medical treatment for sensorineuronal hearing loss caused by the mutation of the deafness-related proteins, including pendrin.

Chiral recognition in the binding of helicenediamine to double strand DNA: interactions between low molecular weight helical compounds and a helical polymer.

Binding of a helicene, 5,8-bis(aminomethyl)-1,12-dimethylbenzo[c]phenanthrene, to calf thymus DNA was studied using UV, CD, and fluorescence spectroscopy as well as calorimetry. The enantiomeric helicenes strongly bound to the double strand DNA possessing the right-handed helical structure. In addition, chiral recognition was observed in the binding, where the (P)-helicene with the right-handed helicity formed more stable complex than the (M)-helicene with the left-handed helicity. The binding studies of the helicenes and natural nucleosides by 1H NMR spectroscopy also revealed the higher affinity to the (P)-helicene. Both monomeric and polymeric nucleic acids thus turned out to favor the (P)-helicity.