Mouse transient receptor potential melastatin 2 (TRPM2) isoform 7 attenuates full-length mouse TRPM2 activity through reductions in its expression by targeting it to ER-associated degradation.

Transient receptor potential melastatin 2 (TRPM2) assembles into tetramers to function as an oxidative stress-sensitive Ca2+ channel at the surface membrane. Limited information is currently available on the 10 protein isoforms of mouse TRPM2 (mTRPM2) identified. This study investigated whether these isoforms function as Ca2+ channels and examined their effects on full-length mTRPM2 activity using the HEK 293 cell exogenous expression system. Only full-length mTRPM2, isoform 1 localized to the surface membrane and was activated by oxidative stress. Isoform 7 was clearly recognized by protein quality control systems and degraded by endoplasmic reticulum-associated degradation after transmembrane proteolysis. In the co-expression system, the activation and expression of full-length mTRPM2 were attenuated by its co-expression with isoform 7, but not with the other isoforms. This decrease in the expression of full-length mTRPM2 was recovered by the proteasomal inhibitor. The present results suggest that isoforms other than isoform 1 did not function as oxidative stress-sensitive channels and also that only isoform 7 attenuated the activation of full-length mTRPM2 by targeting it to endoplasmic reticulum-associated degradation. The present study will provide important information on the functional nature of mTRPM2 isoforms for the elucidation of their roles in physiological and patho-physiological responses in vivo using mouse models.

Antitumor effects of pyrrole-imidazole polyamide modified with alkylating agent on prostate cancer cells.

Androgens and androgen receptor (AR) have a central role in prostate cancer progression by regulating its downstream signaling. Although androgen depletion therapy (ADT) is the primary treatment for most prostate cancers, they acquires resistance to ADT and become castration resistant prostate cancers (CRPC). AR complex formation with multiple transcription factors is important for enhancer activity and transcriptional regulation, which can contribute to cancer progression and resistance to ADT. We previously demonstrated that OCT1 collaborates with AR in prostate cancer, and that a pyrrole-imidazole (PI) polyamide (PIP) targeting OCT1 inhibits cell and castration-resistant tumor growth (Obinata D et al. Oncogene 2016). PIP can bind to DNA non-covalently without a drug delivery system unlike most DNA targeted therapeutics. In the present study, we developed a PIP modified with a DNA alkylating agent, chlorambucil (ChB) (OCT1-PIP-ChB). Then its effect on the growth of prostate cancer LNCaP, 22Rv1, and PC3 cells, pancreatic cancer BxPC3 cells, and colon cancer HCT116 cells, as well as non-cancerous MCF-10A epithelial cells, were analyzed. It was shown that the IC50s of OCT1-PIP-ChB for 22Rv1 and LNCaP were markedly lower compared to other cells, including non-cancerous MCF-10A cells. Comprehensive gene expression analysis of CRPC model 22Rv1 cells treated with IC50 concentrations of OCT1-PIP-ChB revealed that the gene group involved in DNA double-strand break repair was the most enriched among gene sets repressed by OCT1-PIP-ChB treatment. Importantly, in vivo study using 22Rv1 xenografts, we showed that OCT1-PIP-ChB significantly reduced tumor growth compared to the control group without showing obvious adverse effects. Thus, the PIP combined with ChB can exert a significant inhibitory effect on prostate cancer cell proliferation and castration-resistant tumor growth, suggesting a potential role as a therapeutic agent.

Possible involvement of TRPM2 activation in 5-fluorouracil-induced myelosuppression in mice.

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca2+-permeable channel. The activation of TRPM2 by H2O2 causes cell death in various types of cells. 5-Fluorouracil (5-FU) is an important anticancer drug, but myelosuppression is one of the most frequent adverse effects. The involvement of oxidative stress in 5-FU-induced myelosuppression has been reported, and bone marrow cells are known to express TRPM2. The aim of this study was to investigate whether TRPM2 is involved in 5-FU-induced myelosuppression. Enhancement of H2O2-induced intracellular Ca2+ concentration ([Ca2+]i) increase by 5-FU treatment was observed in human embryonic kidney 293 (HEK) cells stably expressing TRPM2 but not in HEK cells, indicating that 5-FU stimulates TRPM2 activation. In CD117 positive cells from wild type (WT) mouse bone marrow, 5-FU also enhanced the H2O2-induced [Ca2+]i increases, but not in cells from Trpm2 knockout (KO) mice. In the CFU-GM colony assay, the 5-FU-induced reduction of colony number was alleviated by Trpm2 deficiency. Moreover, the reduction of leukocytes in blood by administration with 5-FU in WT mice was also alleviated in Trpm2 KO mice. The activation of TRPM2 in bone marrow cells seems to be involved in 5-FU-induced myelosuppression.

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells.

Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.

Short TRPM2 prevents the targeting of full-length TRPM2 to the surface transmembrane by hijacking to ER associated degradation.

Membrane proteins are targeted to the surface transmembrane after folding and assembling in the endoplasmic reticulum (ER). Misfolded- and unassembled-proteins are degraded by proteasomes following ubiquitination, termed ER-associated degradation (ERAD). Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive channel. One of the TRPM2 splicing variants, short TRPM2 (TRPM2-S) having only the N-terminus and first two transmembrane domains, was reported to prevent full-length TRPM2 (TRPM2-L) activation. Although TRPM2-S interacts with TRPM2-L, the inhibitory mechanisms of TRPM2-S are unclear. We found that TRPM2-S prevents transmembrane expression of TRPM2-L by targeting ERAD. TRPM2-S expression was lower than that of TRPM2-L, and was increased by an ERAD inhibitor. TRPM2-S was not expressed at the transmembrane. This suggests that TRPM2-S is a substrate for ERAD. Upon the simultaneous expression of TRPM2-S, the transmembrane expression of TRPM2-L was attenuated and the poly-ubiquitination of TRPM2-L was facilitated. Our study may clarify why TRPM2-S inhibits oxidative stress-induced TRPM2-L activation.

Identification of new octamer transcription factor 1-target genes upregulated in castration-resistant prostate cancer.

Octamer transcription factor 1 (OCT1) is an androgen receptor (AR)-interacting partner and regulates the expression of target genes in prostate cancer cells. However, the function of OCT1 in castration-resistant prostate cancer (CRPC) is not fully understood. In the present study, we used 22Rv1 cells as AR-positive CRPC model cells to analyze the role of OCT1 in CRPC. We showed that OCT1 knockdown suppressed cell proliferation and migration of 22Rv1 cells. Using microarray analysis, we identified four AR and OCT1-target genes, disks large-associated protein 5 (DLGAP5), kinesin family member 15 (KIF15), non-SMC condensin I complex subunit G (NCAPG), and NDC80 kinetochore complex component (NUF2) in 22Rv1 cells. We observed that knockdown of DLGAP5 and NUF2 suppresses growth and migration of 22Rv1 cells. Furthermore, immunohistochemical analysis showed that positive expression of DLGAP5 in prostate cancer specimens is related to poor cancer-specific survival rates of patients. Notably, enhanced expression of DLGAP5 was observed in CRPC tissues of patients. Thus, our findings suggest that these four genes regulated by the AR/OCT1 complex could have an important role in CRPC progression.

Integrative Genomic Analysis of OCT1 Reveals Coordinated Regulation of Androgen Receptor in Advanced Prostate Cancer.

The ligand-dependent transcription factor androgen receptor (AR) plays a critical role in prostate cancer progression. We previously reported that Octamer transcription factor 1 (OCT1), an AR collaborative factor, facilitated the AR genomic bindings to regulate diverse programs of gene expression in AR-dependent prostate cancer cells. Repression of OCT1 binding can serve as a potential treatment strategy for advanced prostate cancer. However, the precise mechanism underlying the functions of OCT1 in advanced prostate cancer, especially lethal castration-resistant prostate cancer (CRPC), is still unclear. To uncover specific OCT1 functions in disease progression, we explored global OCT1-binding regions by performing chromatin immunoprecipitation sequencing in CRPC model 22Rv1 cells. We found that the OCT1 expression level and the obtained OCT1-binding regions increased in 22Rv1 cells compared with AR-dependent prostate cancer LNCaP cells. Interestingly, microarray analysis revealed that OCT1 regulates CRPC-specific target genes in addition to representative AR-regulated genes such as ACSL3. Pathway analysis showed the importance of OCT1 in regulating cell cycle‒related genes. By performing the chromatin immunoprecipitation assay, we validated anillin actin-binding protein (ANLN), which is highly expressed in CRPC and robustly regulated with OCT1 recruitment to the intron and promoter regions in 22Rv1 cells in comparison with LNCaP cells. Furthermore, knockdown of ANLN exhibited impaired cell growth and cell cycle progression, suggesting an important function of ANLN in CRPC cells. In conclusion, these findings raise the possibility that OCT1 coordinates AR signaling in a specific manner that is dependent on disease stage and promotes progression to CRPC.

Protective Effects of Duloxetine against Cerebral Ischemia-Reperfusion Injury via Transient Receptor Potential Melastatin 2 Inhibition.

Activation of transient receptor potential melastatin 2 (TRPM2), an oxidative stress-sensitive Ca2+-permeable channel, contributes to the aggravation of cerebral ischemia-reperfusion (CIR) injury. Recent studies indicated that treatment with the antidepressant duloxetine for 24 hours (long term) attenuates TRPM2 activation in response to oxidative stress in neuronal cells. To examine the direct effects of antidepressants on TRPM2 activation, we examined their short-term (0-30 minutes) treatment effects on H2O2-induced TRPM2 activation in TRPM2-expressing human embryonic kidney 293 cells using the Ca2+ indicator fura-2. Duloxetine exerted the strongest inhibitory effects on TRPM2 activation among the seven antidepressants tested. These inhibitory effects appeared to be due to the inhibition of H2O2-induced TRPM2 activation via an open-channel blocking-like mechanism, because duloxetine reduced the sustained phase but not the initial phase of increases in intracellular Ca2+ concentrations. In a whole-cell patch-clamp study, duloxetine reduced the TRPM2-mediated inward current during the channel opening state. We also examined the effects of duloxetine in a mouse model of CIR injury. The administration of duloxetine to wild-type mice attenuated CIR injury, similar to that in Trpm2 knockout (KO) mice. The administration of duloxetine did not reduce CIR injury further in Trpm2 KO mice, suggesting that it exerts neuroprotective effects against CIR injury by inhibiting TRPM2 activation. Regarding drug repositioning, duloxetine may be a useful drug in reperfusion therapy for ischemic stroke because it has already been used clinically in therapeutics for several disorders, including depression.

Characteristics of the gait adaptation process due to split-belt treadmill walking under a wide range of right-left speed ratios in humans.

The adaptability of human bipedal locomotion has been studied using split-belt treadmill walking. Most of previous studies utilized experimental protocol under remarkably different split ratios (e.g. 1:2, 1:3, or 1:4). While, there is limited research with regard to adaptive process under the small speed ratios. It is important to know the nature of adaptive process under ratio smaller than 1:2, because systematic evaluation of the gait adaptation under small to moderate split ratios would enable us to examine relative contribution of two forms of adaptation (reactive feedback and predictive feedforward control) on gait adaptation. We therefore examined a gait behavior due to on split-belt treadmill adaptation under five belt speed difference conditions (from 1:1.2 to 1:2). Gait parameters related to reactive control (stance time) showed quick adjustments immediately after imposing the split-belt walking in all five speed ratios. Meanwhile, parameters related to predictive control (step length and anterior force) showed a clear pattern of adaptation and subsequent aftereffects except for the 1:1.2 adaptation. Additionally, the 1:1.2 ratio was distinguished from other ratios by cluster analysis based on the relationship between the size of adaptation and the aftereffect. Our findings indicate that the reactive feedback control was involved in all the speed ratios tested and that the extent of reaction was proportionally dependent on the speed ratio of the split-belt. On the contrary, predictive feedforward control was necessary when the ratio of the split-belt was greater. These results enable us to consider how a given split-belt training condition would affect the relative contribution of the two strategies on gait adaptation, which must be considered when developing rehabilitation interventions for stroke patients.

Tyrphostin AG-related compounds attenuate H2O2-induced TRPM2-dependent and -independent cellular responses.

PURPOSE: TRPM2 is a Ca2+-permeable channel that is activated by H2O2. TRPM2-mediated Ca2+ signaling has been implicated in the aggravation of inflammatory diseases. Therefore, the development of TRPM2 inhibitors to prevent the aggravation of these diseases is expected. We recently reported that some Tyrphostin AG-related compounds inhibited the H2O2-induced activation of TRPM2 by scavenging the intracellular hydroxyl radical. In the present study, we examined the effects of AG-related compounds on H2O2-induced cellular responses in human monocytic U937 cells, which functionally express TRPM2. METHODS: The effects of AG-related compounds on H2O2-induced changes in intracellular Ca2+ concentrations, extracellular signal-regulated kinase (ERK) activation, and CXCL8 secretion were assessed using U937 cells. RESULTS: Ca2+ influxes via TRPM2 in response to H2O2 were blocked by AG-related compounds. AG-related compounds also inhibited the H2O2-induced activation of ERK, and subsequent secretion of CXCL8 mediated by TRPM2-dependent and -independent mechanisms. CONCLUSION: Our results show that AG-related compounds inhibit H2O2-induced CXCL8 secretion following ERK activation, which is mediated by TRPM2-dependent and -independent mechanisms in U937 cells. We previously reported that AG-related compounds blocked H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. The inhibitory effects of AG-related compounds on TRPM2-independent responses may be due to scavenging of the hydroxyl radical.

[A CASE OF URETEROUTERINE FISTULA AFTER CAESAREAN SECTION].

The reported incidence rate of iatrogenic ureteral injury is 0.5 to 3% among abdominal surgery. We report a case of ureterouterine fistula after caesarean section. A 38-year-old woman visited our department with a complaint of urinary incontinence without dry time after caesarean section. Several examinations revealed right ureterouterine fistula.Ureteroneocystostomy using psoas hitch and hysterectomy was performed. We found a firm adhesion and stitches around right lower ureter over the uterus, which lead to an additional hysterectomy. After surgery, urinary incontinence had improved. Following two years after surgery, we observed no urinary incontinence or renal dysfunction.

Significance of TRP channels in oxidative stress.

Reactive oxygen species induce oxidative stress, leading to cell damage, but also function as signal transduction molecules. Transient receptor potential (TRP) channels have been attracting increasing attention as Ca2+-permeable channels that sense environmental changes. The members of one class of TRP channels have emerged as reactive oxygen species sensors. The significance of Ca2+ signaling induced by the activation of reactive oxygen species-sensitive TRP channels under pathological conditions is currently being elucidated. The selective inhibition of reactive oxygen species-sensitive TRP channels represents a future challenge that may lead to new therapeutic strategies for the suppression of reactive oxygen species-related diseases.

Targeting TRPM2 in ROS-Coupled Diseases.

Under pathological conditions such as inflammation and ischemia-reperfusion injury large amounts of reactive oxygen species (ROS) are generated which, in return, contribute to the development and exacerbation of disease. The second member of the transient receptor potential (TRP) melastatin subfamily, TRPM2, is a Ca(2+)-permeable non-selective cation channel, activated by ROS in an ADP-ribose mediated fashion. In other words, TRPM2 functions as a transducer that converts oxidative stress into Ca(2+) signaling. There is good evidence that TRPM2 plays an important role in ROS-coupled diseases. For example, in monocytes the influx of Ca(2+) through TRPM2 activated by ROS contributes to the aggravation of inflammation via chemokine production. In this review, the focus is on TRPM2 as a molecular linker between ROS and Ca(2+) signaling in ROS-coupled diseases.

Inhibitory effects of Tyrphostin AG-related compounds on oxidative stress-sensitive transient receptor potential channel activation.

Some transient receptor potential (TRP) proteins including TRPA1, TPRM2 and TRPV1 are oxidative stress-sensitive Ca(2+)-permeable channels. Ca(2+) signaling via these TRP channels activated by oxidative stress has been implicated in the aggravation of various inflammatory diseases and pain sensation. We recently reported that Tyrphostin AG490 exerted inhibitory effects on H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. In order to identify stronger inhibitors of oxidative stress-sensitive TRP channels than AG490, we examined the inhibitory effects of Tyrphostin AG-related compounds on H2O2-induced TRP channel activation in human embryonic kidney 293 cells expressing TRP channels. AG555 and AG556 blocked the activation of TRPM2 by H2O2 more strongly than AG490. Regarding TRPV1 and TRPA1, none of the three compounds tested affected H2O2-induced TRPV1 activation; however, AG555 and AG556 reduced H2O2-induced TRPA1 activation more than AG490. Thus, we herein identified AG555 and AG556 as new compounds that exert stronger inhibitory effects on H2O2-induced TRPM2 and TRPA1 activation than AG490. Edaravone, a hydroxyl radical scavenger used in the treatment of cerebral hemorrhage and cerebral infarction, did not affect H2O2-induced TRPM2 or TRPA1 activation. AG555 and AG556 may be useful seed compounds as therapeutic agents for several TRP-related diseases associated with oxidative stress.

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation.

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation.

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2 channels by Fe(2+) may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe(2+) is released by heme degradation under intracerebral hemorrhage.

Rhythmic arm swing enhances patterned locomotor-like muscle activity in passively moved lower extremities.

The use of driven gait orthosis (DGO) has drawn attention in gait rehabilitation for patients after central nervous system (CNS) lesions. By imposing a passive locomotor-like kinematic pattern, the neural mechanisms responsible for locomotion can be activated as in a normal gait. To further enhance this activity, discussions on possible intervention are necessary. Given the possible functional linkages between the upper and lower limbs, we investigated in healthy subjects the degree of modification in the lower limb muscles during DGO-induced passive gait by the addition of swing movement in the upper extremity. The results clearly showed that muscle activity in the ankle dorsiflexor TA muscle was significantly enhanced when the passive locomotor-like movement was accompanied by arm swing movement. The modifications in the TA activity were not a general increase through the stride cycles, but were observed under particular phases as in normal gaits. Voluntary effort to swing the arms may have certain effects on the modification of the muscle activity. The results provide clinical implications regarding the usefulness of voluntary arm swing movement as a possible intervention in passive gait training using DGO, since ordinary gait training using DGO does not induce spontaneous arm swing movement despite its known influence on the lower limb movement.

Prevention of aberrant protein aggregation by anchoring the molecular chaperone αB-crystallin to the endoplasmic reticulum.

The chaperone αB-crystallin (αBC) is a member of the small heat shock protein family and its point or truncated mutants cause the muscular disorder α-crystallinopathy. The illness is histologically characterized by accumulation of protein aggregates in muscle cells. Expression of the myopathy-causing R120G mutant of αBC, harboring an arginine-to-glycine mutation at position 120, results in aggregate formation. We demonstrated that tethering αBC to the endoplasmic reticulum (ER) membrane represses the protein aggregation mediated by the R120G mutant. ER-anchored αBC decreased the amount of the R120G mutant through autophagic proteolysis. In contrast, knockdown of ATG5, an E3 ligase essential for autophagy, in ER-anchored αBC-transfected cells restored the quantity of the R120G mutant. In this context, aggregate formation was still suppressed, indicating that ER-anchored αBC profoundly constrains aggregation competency of the R120G mutant separately from downregulating the abundance of the mutant. We have proposed that protein aggregation is prevented by manipulation of the ER microenvironment with αBC, and have shed light on a novel aspect of the ER as a therapeutic target.

Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity.

Calumin is an endoplasmic reticulum (ER)-transmembrane protein, and little is known about its physiological roles. Here we showed that calumin homozygous mutant embryos die at embryonic days (E) 10.5-11.5. At mid-gestation, calumin was expressed predominantly in the yolk sac. Apoptosis was enhanced in calumin homozygous mutant yolk sacs at E9.5, pointing to a possible link to the embryonic lethality. Calumin co-immunoprecipitated with ERAD components such as p97, BIP, derlin-1, derlin-2 and VIMP, suggesting its involvement in ERAD. Indeed, calumin knockdown in HEK 293 cells resulted in ERAD being less efficient, as demonstrated by attenuation in both degradations of a misfolded α1-antitrypsin variant and the ER-to-cytosol dislocation of cholera toxin A1 subunit. In calumin homozygous mutant yolk sac endoderm cells, ER stress-associated alterations were observed, including lipid droplet accumulation, fragmentation of the ER and dissociation of ribosomes from the ER. In this context, the ER-overload response, assumed to be cytoprotective, was also triggered in the mutant endoderm cells, but seemed to fully counteract the excessive ER stress generated due to defective ERAD. Taken together, our findings suggested that calumin serves to maintain the yolk sac integrity through participation in the ERAD activity, contributing to embryonic development.

Anti-phase action between the angular accelerations of trunk and leg is reduced in the elderly.

Quiet standing posture in humans has often been modeled as a single inverted pendulum pivoting around the ankle joint. However, recent studies have suggested that anti-phase action between leg and trunk segments plays a significant role in stabilizing posture by reducing the acceleration of the center of mass (COM) of the body. The aim of this study is to test the hypothesis that anti-phase action is attenuated in the elderly compared to the young. The anterior-posterior movements of leg and trunk segments were measured using 4 laser displacement sensors from 22 healthy young subjects (age range, 20-35 years) and 38 healthy elderly subjects (age range, 57-80 years) standing quietly for 30s twice. To focus on the segmental action between trunk and legs, we applied constraints (i.e., wooden splints) on each segment. We found that the velocity and acceleration of the COM (standard deviation of the time series was evaluated) were significantly higher for the elderly subjects than for young subjects. The increase in the acceleration of the COM resulted not only from an increase in the angular acceleration of the segments but also from the reduction of their anti-phase relationship, as demonstrated by an index that quantifies the degree of cancelation between both segments. We conclude that the degree of anti-phase action between trunk and leg segments during quiet standing is smaller for elderly subjects than for young subjects, and that this change of the anti-phase action due to aging resulted in increased COM acceleration in the elderly subjects.