Shin Aizawa

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-κB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-κB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

Substance P-induced cadherin expression and its signal transduction in a cloned human corneal epithelial cell line

Although the absence of Substance P (SP), a neurotransmitter in the trigeminal nerve, has been speculated as a cause for developing neurotrophic keratitis, its exact pathogenesis is still not clarified. In a previous report, we showed with electron microscopic examination that epithelial cell attachment was weakened in denervated corneas. In this study, SV40‐transformed human corneal epithelial cells (HCE‐Ts) were used to explore the molecular mechanisms responsible for mediating regulation of E‐cadherin expression in response to Substance P receptor stimulation. Expression of the mRNAs for specific SP receptors, neurokinin (NK)‐1R, NK‐2R, and NK‐3R, was demonstrated with RT‐PCR. The cells were treated with various concentrations of SP in vitro, and the expression of an adhesion molecule E‐cadherin was analyzed by immunofluorescence, immunoblotting, and enzyme‐linked immunosorbent assay (ELISA) using an anti‐E‐cadherin antibody. E‐cadherin expression was increased by SP in a dose‐dependent manner both in the cytosolic fraction and in the cell membrane fraction. This increase in E‐cadherin expression was completely inhibited by Calphostin C (PKC inhibitor) and KN‐62 (CaMK inhibitor), but not by H‐89 (PKA inhibitor), indicating that SP‐induced E‐cadherin expression involves the activation of protein kinase C (PKC) and calmodulin kinase (CaMK). SP did not affect cell proliferation at all. All these findings indicate that SP induced E‐cadherin expression through PKC and CaMK activation and suggest that a lack of SP may account in part for the pathogenesis of neurotrophic keratitis. J. Cell. Physiol. 182:189–195, 2000.

Senescent B lymphopoiesis is balanced in suppressive homeostasis: Decrease in interleukin-7 and transforming growth factor-β levels in stromal cells of senescence-accelerated mice

The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-β (TGF-β) was found to contribute to B cell suppression. To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-β during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7–responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-β is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-β antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 μg/ml). Furthermore, TGF-β protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-β concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-β activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosupression).

Mutations in Flk, FlgG, FlhA, and FlhE That Affect the Flagellar Type III Secretion Specificity Switch in Salmonella enterica

Upon completion of the flagellar hook-basal body (HBB) structure, the flagellar type III secretion system switches from secreting rod/hook-type to filament-type substrates. The secretion specificity switch has been reported to occur prematurely (prior to HBB completion) in flk-null mutants (P. Aldridge, J. E. Karlinsey, E. Becker, F. F. Chevance, and K. T. Hughes, Mol. Microbiol. 60:630-643, 2006) and in distal rod gene gain-of-function mutants (flgG* mutants) that produce filamentous rod structures (F. F. Chevance, N. Takahashi, J. E. Karlinsey, J. Gnerer, T. Hirano, R. Samudrala, S. Aizawa, and K. T. Hughes, Genes Dev. 21:2326-2335, 2007). A fusion of beta-lactamase (Bla) to the C terminus of the filament-type secretion substrate FlgM was used to select for mutants that would secrete FlgM-Bla into the periplasmic space and show ampicillin resistance (Ap(r)). Ap(r) resulted from null mutations in the flhE gene, C-terminal truncation mutations in the flhA gene, null and dominant mutations in the flk gene, and flgG* mutations. All mutant classes required the hook length control protein (FliK) and the rod cap protein (FlgJ) for the secretion specificity switch to occur. However, neither the hook (FlgE) nor the hook cap (FlgD) protein was required for premature FlgM-Bla secretion in the flgG* and flk mutant strains, but it was in the flhE mutants. Unexpectedly, when deletions of either flgE or flgD were introduced into flgG* mutant strains, filaments were able to grow directly on the filamentous rod structures.

Rose odor can innately counteract predator odor

When animals smell a predator odor such as 2,5-Dihydro-2,4,5-trimethylthiazoline (TMT), even if it is a novel substance, the hypothalamo-pituitary-adrenal (HPA) axis is activated, causing stress-like behaviors. Although the medial part of the bed nucleus of stria terminalis (mBST) is known to be involved in this process, the mechanism remains unclear. Moreover, it is unknown whether there is any odor that can counteract the predator odor, even when the odorants are novel substances for the animals. In this study, we assessed whether rose odor can counteract by counting the number of activated neurons in mice brain following the presentation of rose odor with or without TMT for 30 min. The number of activated cells in the mBST and in the ventrorostral part of the anterior piriform cortex (APC) was significantly reduced by a mixture of TMT and rose odor; however, no significant differences were noted in the dorsal part of the APC and in the olfactory bulb (OB) following TMT presentation with or without rose odor. The results suggest that rose odor may counteract the TMT-induced stress response in the OB and/or APC and suppress the neural circuit to the mBST. It also indicates that there are some odors that can innately counteract predator odor, even when they have not been experienced before.

Decreased Proliferation and Erythroid Differentiation of K562 Cells by siRNA-induced Depression of OCTN1 (SLC22A4) Transporter Gene

PurposeRecently, it was reported that OCTN1 transporter (SLC22A4) is associated with rheumatoid arthritis (RA) and Crohn’s disease. Additionally, we reported that OCTN1 is expressed in hematopoietic cells, preferentially in erythroid cells. Accordingly, we assessed the physiological role of OCTN1 by examining the effect of knockdown of OCTN1 in blood cells using siRNA method.Materials and MethodsVector-based short hairpin RNA (shRNA) was used to establish K562 cell line which shows stably decreased expression of OCTN1. The characteristic of knockdown of OCTN1 in K562 cells was investigated by cell proliferation, cell differentiation, and uptake of ergothioneine that is a good substrate of OCTN1.Results Several clones of K562 cells exhibited significantly reduced expression of OCTN1 mRNA and protein. They also showed a decreased growth rate and butyrate-dependent differentiation to erythrocytes compared with control-vector transfected cells. In addition, uptake of [3H]ergothioneine by K562 cells suggested that Na+-dependent and high-affinity transporter which is similar to the characteristics of OCTN1 is functional. Moreover, uptake of ergothioneine by K562 cells which exhibit decreased-expression of OCTN1 was decreased in comparison with wild type K562 cells.ConclusionsIt was suggested that OCTN1 is involved in the transport of physiological compounds that are important for cell proliferation and erythroid differentiation.

Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters: A plausible gateway for the salvage pathway of Tetrahydrobiopterin biosynthesis

Tetrahydrobiopterin (BH(4)) is synthesized de novo in particular cells, but in the case of a systemic or local BH(4) deficiency, BH(4) supplementation therapy is applied. BH(4)-responsive PKU has also been effectively treated with BH(4) supplementation. However, the rapid clearance of the supplemented BH(4) has prevented the therapy from being widely accepted. Deposition of BH(4) after supplementation involves oxidation of BH(4) to dihydrobiopterin (BH(2)) and subsequent conversion to BH(4) by the salvage pathway. This pathway is known to be almost ubiquitous in the body. However, the mechanism for the redistribution and exclusion of BH(4) across the plasma membrane remains unclear. The aim of this work was to search for the key transporter of the uptake precursor of the salvage pathway. Based on the observed sensitivity of pterin transport to nitrobenzylthioinosine (NBMPR), we examined the ability of ENT1 and ENT2, representative equilibrative nucleoside transporters, to transport sepiapterin (SP), BH(2) or BH(4) using HeLa cell and Xenopus oocyte expression systems. hENT2 was capable of transporting the pterins with an efficiency of SP>BH(2)>BH(4). hENT1 could also transport the pterins but less efficiently. Non-transfected HeLa cells and rat aortic endothelial cells were able to incorporate the pterins and accumulate BH(4) via uptake that is likely mediated by ENT2 (SP>BH(2)>BH(4)). When exogenous BH(2) was given to mice, it was efficiently converted to BH(4) and its tissue deposition was similar to that of sepiapterin as reported (Sawabe et al., 2004). BH(4) deposition after BH(2) administration was influenced by prior treatment with NBMPR, suggesting that the distribution of the administered BH(2) was largely mediated by ENT2, although urinary excretion appeared to be managed by other mechanisms. The molecular basis of the transport of SP, BH(2), and BH(4) across the plasma membrane has now been described for the first time: ENT2 is a transporter of these pterins and is a plausible gateway to the salvage pathway of BH(4) biosynthesis, at least under conditions of exogenous pterin supplementation. The significance of the gateway was discussed in terms of BH(2) uptake for BH(4) accumulation and the release for modifying the intracellular BH(2)/BH(4) ratio.

Inflammatory Biomarker, Neopterin, Suppresses B Lymphopoiesis for Possible Facilitation of Granulocyte Responses, Which Is Severely Altered in Age-Related Stromal-Cell–Impaired Mice, SCI/SAM

Neopterin is produced by monocytes and is a useful biomarker of inflammatory activation. We found that neopterin enhanced in vivo and in vitro granulopoiesis triggered by the stromal-cell production of cytokines in mice. The effects of neopterin on B lymphopoiesis during the enhancement of granulopoiesis were determined using the mouse model of senescent stromal-cell impairment (SCI), a subline of senescence-accelerated mice (SAM). In non-SCI mice (a less senescent stage of SCI mice), treatment with neopterin decreased the number of colonies, on a semisolid medium, of colony-forming units of pre–B-cell progenitors (CFU-preB) from unfractionated bone marrow (BM) cells, but not that from a population rich in pro-B and pre-B cells without stromal cells. Neopterin upregulated the expression of genes for the negative regulators of B lymphopoiesis such as tumor necrosis factor-α (TNF-α ), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β) in cultured stromal cells, implying that neopterin suppressed the CFU-preB colony formation by inducing negative regulators from stromal cells. The intraperitoneal injection of neopterin into non-SCI mice resulted in a marked decrease in the number of femoral CFU-preB within 1 day, along with increases in TNF-α and IL-6 expression levels. However, in SCI mice, in vivo and in vitro responses to B lymphopoiesis and the upregulation of cytokines after neopterin treatment were less marked than those in non-SCI mice. These results suggest that neopterin predominantly suppressed lymphopoiesis by inducing the production of negative regulators of B lymphopoiesis by stromal cells, resulting in the selective suppression of in vivo B lymphopoiesis. These results also suggest that neopterin facilitated granulopoiesis in BM by suppressing B lymphopoiesis, thereby contributing to the potentiation of the inflammatory process; interestingly, such neopterin function became impaired during senescence because of attenuated stromal-cell function, resulting in the downmodulation of the host-defense mechanism in the aged.

Therapeutic effect of Photodynamic therapy using Na-Pheophorbide a on osteomyelitis models in rats

OBJECTIVE In this study, we examined the therapeutic effect of photodynamic therapy (PDT) using the photosensitizer Na-Pheophorbide a (Na-Phde a) on osteomyelitis models in rats. BACKGROUND Osteomyelitis is one of the most serious infectious problems in the orthopedic field. Recently, as a new clinical approach against septic arthritis, an experimental in vivo and in vitro model for the inactivation of methicillin-resistant-Staphylococcus aureus by PDT using Na-Phde a has been developed. METHODS Methicillin-sensitive Staphylococcus aureus (MSSA) was injected into the tibia of the rats to create osteomyelitis models (n = 10, 10 legs). A total of 560 μmol/l of Na-Phde a solution was injected into five of these tibial osteomyelitis models (five legs) 48 h after the initial MSSA infection. Sixty minutes after the Na-Phde a injection, a semiconductor laser (125 mW, 670 nm) was used to irradiate the models for 10 min with a total energy of 93.8 J/mm(2). As a control group, five rats (five legs) were treated with a phosphate buffered saline injection at 48 h after MSSA infection. Weight and leg perimeter changes were plotted. Bacterial growth, histological examination and radiological examination were evaluated at 14 days after initial treatment. RESULTS PDT with Na-Phde a significantly prevented leg swelling. In the PDT group, bone destruction owing to osteomyelitis was inhibited not only histologically but also radiographically. CONCLUSIONS The results in these experiments show that PDT using Na-Phde a improved osteomyelitis in rats. This suggests that PDT using Na- Phde a can be a useful treatment for osteomyelitis.