Analysis of the risk factors of mortality in elderly patients with hip fracture using a combined database of medical and long-term care insurance claims data.

BACKGROUND: The present study evaluated the risk of mortality in elderly hip fracture, focusing on comorbidities and nursing care levels. METHODS: The present study was an observational cohort study that used a combined database of medical and long-term care insurance (LTCI) claims data from one prefecture in Japan between 2011 and 2016. In total, 6125 patients aged 65 years and older were selected from acute care hospitals with a diagnosis of "hip fracture" between March 2011 and March 2012. The impact of long-term care insurance claim evaluation levels and comorbidities at recruitment time was investigated using this dataset. These patients were followed up monthly until March 2016. Based on this person-month dataset, survival analysis was performed with death as the endpoint. Cases in which receipt data were missing during the middle of the observation period and cases in which the patient survived at the end of the observation period were censored. RESULTS: The number of deaths during the observation period was 635 (10.4%). The impact of comorbidities and nursing care level on mortality were both significant as follows: high nursing care level before the fracture (hazard ratio: 1.09, P < 0.001), comorbidities of malignant diseases (HR: 1.45, P < 0.001), heart disease (hazard ratio: 1.20, P = 0.037), pneumonia (hazard ratio: 1.27, P < 0.001), chronic obstructive pulmonary disease (hazard ratio: 1.28, P = 0.026), renal failure (hazard ratio: 1.44, P < 0.001), and dementia (hazard ratio: 1.27, P = 0.013). CONCLUSION: The results of this study showed that a high level of nursing care and presence of comorbidities such as malignant diseases, heart diseases, pneumonia, chronic obstructive pulmonary disease, renal failure, and dementia increased mortality in elderly patients with hip fracture. Furthermore, this study showed the usefulness of a combined database of medical and LTCI claims data for clinical and health service-related research in the field of orthopedics.

Decline incidence in upper gastrointestinal bleeding in several recent years: data of the Japan claims database of 13 million accumulated patients.

This study was to examine the recent trends in upper gastrointestinal bleeding in Japan using a large-scale real-world database. The incidence of upper gastrointestinal bleeding was evaluated in the Japan Medical Data Center claims database of 13,019,713 patients aged 20 to 74 years with traceability for 3 months from 2009 to 2014. The incidence was compared with peptic ulcers and gastroesophageal reflux disease. The prescription of medications was also evaluated. The incidence of bleeding was 0.137%, 0.121%, 0.113%, 0.106%, 0.099%, and 0.105% during 2009 to 2014 with a time-dependent decline (p<0.001). Peptic ulcers (>10 times higher than the incidence of bleeding) decreased with time (p<0.001), whereas gastroesophageal reflux disease increased (p = 0.006). Upper gastrointestinal bleeding was higher in male patients and older patients (60-74 years old) (p<0.001 respectively). The prescription rate of antithrombotic medications and proton pump inhibitors increased from 2009 to 2014 (p<0.001 respectively). The incidence of upper gastrointestinal bleeding decreased from 2009 to 2014 in this relatively large-scale real-world database in Japan, concomitant with the decrease in peptic ulcers. The decreased incidence might have been due to changes in the disease structure and therapeutic strategies over time.

Risk for prolonged hospitalization and mortality in aged community acquired pneumonia patients: a retrospective study in Japan.

The present study aimed to reveal; i) risk for prolonged hospitalization and mortality in aged community acquired pneumonia patients, and ii) whether swallowing ability was related to re-hospitalization. The present retrospective study included 92 patients older than 75 years hospitalized with community acquired pneumonia in Takagi Hospital between April 2017 and March 2018. The patients were classified into 3 groups; discharged within 17 days (group I): hospitalized more than 18 days (group II): died during the hospitalization (group III). Swallowing ability was evaluated if available. Univariate analysis indicated males and body mass index (BMI) in group I (n = 24) were higher than group II (n = 46). Group III (n = 22) had low serum albumin, low BMI, and severe disease progression compared with group I. Multivariate analysis demonstrated that group II BMI was lower than group I [odds ratio (OR) = 1.18, p = 0.042]. Group III had lower serum albumin level compared with group I (OR = 81.01, p = 0.025). Diabetes mellitus (p = 0.009), but not swallowing disability, was risk for readmission. Malnutrition represented by low albumin enhanced mortality rate in the pneumonia patients, and low BMI and diabetes mellitus might increase the pneumonia risk.

Possibility of using salivary ultra-weak chemiluminescence as a biomarker for feelings of anxiety in hospital settings.

The aim of this study was to assess whether a particular value of noninvasive salivary ultra-weak chemiluminescence (UCL) could be used as a biomarker of psychological stress. Our study covered two groups. Group 1 comprised six healthy volunteers who stayed in a hospital for one night and group 2 comprised 15 patients with lung cancer and 24 patients with respiratory diseases other than lung cancer who were in hospital for an extended stay. First, we evaluated the UCL of saliva from six healthy volunteers before and after one night in hospital. Immunoglobulin A (IgA) concentrations were also measured. The integrated intensity value of UCL was correlated with the IgA concentration (correlation coefficient 0.90). Second, in the case of a long hospital stay, we found that the maximum salivary UCL intensities were higher in patients with lung cancer than in those with respiratory diseases other than lung cancer or in 28 healthy controls. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a saliva collection device for use in the field.

BACKGROUND: For monitoring biomarkers, saliva has several distinct advantages over other biological fluids. Saliva sampling is relatively non-invasive and enables the collection from either adults or infants under many different circumstances. However, there is no collection device that can be speedily used for analysis in the field. The aim of the present study was to evaluate the suitability of a new device, termed Muddler, compared with commercially available collection devices. METHODS: Saliva was collected from healthy volunteers. The collection devices such as Muddler, eye sponge, Salivette® Cotton (SC) and Salivette® Synthetic (SS) were evaluated in terms of the volume and/or composition of the collected saliva. The amounts of immunoglobulin A (IgA) and lactofferin in saliva were assessed by the enzyme-linked immunosorbent assays with the corresponding antibodies. Amylase activity was measured using a commercially available kit, and high molecular weight complexes including mucin were assessed by SDS-PAGE staining. RESULTS: A newly developed Muddler, which was made of plastic plate, was the best device for collecting a constant volume of saliva among all the devices examined in the present study. Furthermore, Muddler can collect without change in composition of salivary proteins such as IgA, lactoferrin, amylase, and mucin complex, whereas the levels of the salivary proteins obtained with all the commercial devices used were clearly different from those in original saliva. CONCLUSIONS: The newly developed Muddler was the best collection device in terms of the accuracy of collection and the reliability of measurements among all the devices examined in the present study.

Ribosome-small-subunit-dependent GTPase interacts with tRNA-binding sites on the ribosome.

RsgA (ribosome-small-subunit-dependent GTPase A, also known as YjeQ) is a unique GTPase in that guanosine triphosphate hydrolytic activity is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome, and the maturation of 16S rRNA. To study the interaction of Escherichia coli RsgA with the ribosome, chemical modifications using dimethylsulfate and kethoxal were performed on the small subunit in the presence or in the absence of RsgA. The chemical reactivities at G530, A790, G925, G926, G966, C1054, G1339, G1405, A1413, and A1493 in 16S rRNA were reduced, while those at A532, A923, G1392, A1408, A1468, and A1483 were enhanced, by the addition of RsgA, together with 5'-guanylylimidodiphosphate. Among them, the chemical reactivities at A532, A790, A923, G925, G926, C1054, G1392, A1413, A1468, A1483, and A1493 were not changed when RsgA was added together with GDP. These results indicate that the binding of RsgA induces conformational changes around the A site, P site, and helix 44, and that guanosine triphosphate hydrolysis induces partial conformational restoration, especially in the head, to dissociate RsgA from the small subunit. RsgA has the capacity to coexist with mRNA in the ribosome while it promotes dissociation of tRNA from the ribosome.

Responses of ultra-weak chemiluminescence and secretory IgA in saliva to the induction of angry and depressive moods.

Oxidative damage to tissues and cells contributes to disease processes. We used ultra-weak chemiluminescence (uwCL) as an indicator of oxidative activity to examine the effects of psychological challenges on oxidative responses. We also examined the association of underlying psychological characteristics with oxidative and immune responses. Eighteen healthy men and women with a mean age of 24.1 were recruited. Anger and depressive symptoms were evaluated using the State-Trait Anger Expression Inventory and the Center for Epidemiological Studies Depression Scale, respectively. Following a baseline period, participants were required to complete two separate speech tasks where they were asked to recall life events that made them feel angry (AT) or depressed (DT). The tasks were separated by a 30-min recovery period and the order was randomized between participants using a counterbalanced design. Saliva was sampled and assayed for uwCL and secretory immunoglobulin A (sIgA). The level of uwCL was significantly increased in response to both tasks (p<.05), whereas sIgA concentrations decreased significantly in response to DT (p<.05). At 30 min after each task, uwCL values were positively related to anger-in (p<.005), anger expression (p<.05) and trait anger (p<.05) post-AT, and sIgA concentrations were positively related to anger-out (p<.05) post-AT and -DT, after controlling for covariates. The present study suggests that induction of angry and depressive moods can increase oxidative activity and transiently weaken immunity indicated by salivary sIgA concentrations. In addition, anger personality traits may modify these responses.

Interaction between RsgA and the ribosome.

RsgA is a unique GTP hydrolytic protein that is widely found in bacteria and plants, and is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome in cells and maturation of 16S ribosomal RNA. Here, we investigated the interaction between EscherichiacoliRsgA and the ribosome. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA, suggesting that RsgA binds to the decoding center. Chemical footprinting was also performed to further investigate the interaction.

Comparison of peroxidase response to mental arithmetic stress in saliva of smokers and non-smokers.

Saliva is the first body fluid to encounter exogenous materials or gases such as cigarette smoke (CS). The aim of this study was to examine whether smoking affects oral peroxidase (OPO) reactivity to mental stress. The subjects were 39 non-smokers and 10 smokers. In the experiment, the Kraepelin psychodiagnostic test as a psychological stressor and saliva was sampled 30 min before, just before, immediately after, and 30 min after the beginning of the test. OPO reactivity to the test between smokers and non-smokers was measured in addition to uric acid concentration, flow rate, IgA, thiocyanate (SCN-) concentration, amylase activity as a salivary stress marker, and ultra-weak chemiluminescence (UCL) level, which is indicative of salivary antioxidative and antibacterial abilities. Moreover, we studied the effect of smoking on the response of salivary peroxidase (SPO) and myeloperoxidase (MPO) activity to mental stress, respectively. The results showed that the IgA concentration, amylase activity, SCN(- concentration, and UCL level are higher in the non-smoking group than smoking group and the IgA concentration and UCL level increased in the non-smokers significantly just after the Kraepelin test. The levels of SCN-) were higher in smokers than in non-smokers and OPO activity was greater in the non-smoking group in all sessions. Furthermore, only the non-smokers had significantly increased MPO activity just after the test. MPO may play a crucial role in the response to acute psychological stress besides inflammation, and CS suppresses this response significantly.

Angiotensin II receptor blockade ameliorates mesangioproliferative glomerulonephritis in rats through suppression of CTGF and PAI-1, independently of the coagulation system.

BACKGROUND: Previously we observed that the coagulation system promotes matrix protein accumulation through transforming growth factor (TGF)-beta and connective tissue growth factor (CTGF) expression in rat mesangioproliferative glomerulonephritis (MsPGN). Angiotensin II receptor blockers (ARBs) are known to suppress matrix accumulation in experimental MsPGN. In the present study, we investigated whether ARB suppresses MsPGN through inhibition of these profibrotic cytokines, and in relation to coagulation and fibrinolytic systems. METHODS: MsPGN was induced in Wistar rats by intravenous injection of anti-Thy-1.1 monoclonal antibody, OX-7. As an ARB, olmesartan was orally administered in rat feed from the day of OX-7 injection (day 0) to day 8, when rats were sacrificed and kidney specimens were collected. The degrees of cellular proliferation, matrix production, coagulation factors, and inhibitory factor of fibrinolysis were evaluated. RESULTS: Although blood pressure did not change in the normal, disease control, or treatment groups, the amount of urinary protein was significantly decreased in the ARB-treated groups, compared with the disease control group (p < 0.05). alpha-Smooth muscle actin expression was suppressed significantly in the treatment groups (p < 0.001). Blue-staining areas of trichrome, the number of proliferating cell nuclear antigen (PCNA)- or ED-1-positive cells, fibronectin and plasminogen activator inhibitor type 1 in glomeruli significantly decreased in the treatment groups (p < 0.05, respectively); however, fibrin-related antigen and factor V depositions were not suppressed in the treatment groups. CONCLUSIONS: These results suggest that the ARB drug would ameliorate MsPGN in vivo, at least partly through CTGF and plasminogen activator inhibitor type 1 suppression, and independently of the local coagulation system in glomeruli.

Dysfunction of paracellin-1 by dephosphorylation in Dahl salt-sensitive hypertensive rats.

A high-salt diet reduced the levels of renal cAMP content and serine-phosphorylated paracellin-1 in Dahl salt-sensitive hypertensive rats. In MDCK cells expressing paracellin-1, protein kinase A inhibitor reduced the serine-phosphorylated paracellin-1 and transepithelial Mg(2+) transport, suggesting that a dephosphorylation of paracellin-1 induces the reduction of Mg(2+) reabsorption in salt-sensitive hypertension.

P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways.

Extracellular ATP causes apoptosis and/or necrosis of the hemopoietic lineage through the activation of P2X7 receptors. In this study, we investigated P2X7 receptor-mediated cell death during murine T cell maturation. The expression level and activity of P2X7 receptors, as measured by induction of cell death and pore formation, were higher in splenocytes than thymocytes. Flow cytometric analysis revealed that cell shrinkage was induced by activation of the P2X7 receptor in murine lymphocytes and the responding cells were T cells. Splenic T cells were more responsive than their thymic counterpart. These observations indicate that the system of P2X7 receptor-mediated cell death in T cells could be modulated during T cell maturation. Furthermore, decreased extracellular Cl- suppressed ATP-induced cell shrinkage in splenocytes without inhibiting ERK1/2 phosphorylation, which is reported to mediate necrotic cell death. Treatment with U0126 (a MEK inhibitor) suppressed ATP-induced ERK1/2 phosphorylation without inhibiting cell shrinkage. Moreover, decreased extracellular Cl- and treatment with U0126 suppressed ATP-induced cell death. These observations indicate that the activation of P2X7 receptor leads to T cell death by two independent pathways, one of which is cell shrinkage dependent and the other of which involves the phosphorylation of ERK1/2. In conclusion, we demonstrate increasing P2X7 receptor activity during T cell maturation and the existence of two essential pathways in P2X7 receptor-mediated T cell death. Our findings suggest that ATP-induced cell death of peripheral T lymphocytes is important in P2X7 receptor-regulated immune responses.

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions.

Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2',5'-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells.

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK(1) cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.

Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells.

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [14C]-alpha-methyl glucopyranoside ([14C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [14C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.

Involvement of chloride in apoptotic cell death induced by activation of ATP-sensitive P2X7 purinoceptor.

The ATP-gated P2X(7) receptor is a plasma membrane receptor belonging to the family of P2X purinoceptors. Its activation leads to multiple downstream events including influx of ions, pore formation to allow the passage of larger molecular weight species, and cell death by apoptosis and/or necrosis. The cell death is thought to be correlated with the pore formation but does not directly result from the dilatation of pores. We have generated and characterized a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor. In this study, we investigated the mechanism of P2X(7) receptor-induced cell death using this clone. Treatment with P2X(7) receptor agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP induced depolarization of membrane potential, pore formation, and cell shrinkage, an early hallmark of apoptosis in the buffer containing physiological concentrations of ions. Analysis by flow cytometry revealed that the activity of pore formation in shrunk cells was much higher than in non-shrunk cells. The activation of P2X(7) receptor also caused the release of lactate dehydrogenase from cells. The P2X(7) receptor-mediated cell shrinkage and lactate dehydrogenase release were blocked when media Cl(-) was replaced with gluconate. However, removal of extracellular Cl(-) did not affect plasma membrane depolarization and pore formation by treatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP. Therefore we concluded that pore formation plays a critical role in the P2X(7) receptor-induced apoptotic cell death and that this is mediated by extracellular Cl(-) influx.

Reorganization of ZO-1 by sodium-dependent glucose transporter activation after heat stress in LLC-PK1 cells.

Heat stress (HS) induces activation of high-affinity sodium-dependent glucose transporter (SGLT1) in porcine renal LLC-PK(1) cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens-1 (ZO-1), a cytosolic tight junction (TJ) protein, after HS. HS (42 degrees C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37 degrees C for 12 h increased TER above pre-HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC-dextran (4,000 Da). Immunofluorescent staining of ZO-1 showed that HS diffused ZO-1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO-1 was distributed from the Triton X-100 insoluble to the Triton X-100 soluble pool. After incubation at 37 degrees C for 12 h, cell contact and ZO-1 extractability with Triton X-100 returned to pre-HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC-dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO-1 extractability with Triton X-100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO-1 mediated by elevation of tyrosine kinases activity after heat injury.

Association of paracellin-1 with ZO-1 augments the reabsorption of divalent cations in renal epithelial cells.

Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.

A novel GTPase activated by the small subunit of ribosome.

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.

Arachidonic acid-activated Na+-dependent Mg2+ efflux in rat renal epithelial cells.

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg2+ deficiency. We examined whether the extracellular Mg2+ concentration affects AA production and whether AA regulates a putative Na+-dependent Mg2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg2+-containing medium for 2 days because they enable us to detect Na+-stimulated Mg2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg2+ increased AA release both in the absence and presence of extracellular Na+. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 microM), an inhibitor of cytosolic phospholipase A) (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2), and bromoenol lactone (BEL, 10 microM), an inhibitor of iPLA2. However, LY-311727 (10 microM), a secretory phospholipase A2 (sPLA2) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA2 and iPLA2 mRNAs, but not sPLA2. In the mag-fura 2 fluorescence measurements, extracellular Mg2+ removal caused slight decrease in the intracellular free Mg2+ concentration ([Mg2+]i) in the Na+-free condition. The addition of Na+ caused a rapid decrease in [Mg2+]i, indicating the presence of a Na+-dependent Mg2+ efflux pathway. The Na+-dependent [Mg2+]i decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 microM), indomethacin (10 microM) and 17-octadecynoic acid (ODYA) (10 microM), enhanced the Na+-dependent [Mg2+]i decrease. Furthermore, the addition of exogenous AA (30 microM) enhanced the Na+-dependent [Mg2+]i decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na+/Mg2+-exchanger inhibitor. These results suggest that extracellular Mg2+ removal elevates AA release mediated mainly by iPLA2 and that AA upregulates the Na+-dependent Mg2+ efflux in NRK-52E cells.