Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca2+.

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6's physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich-Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca(2+) is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium.

The role of TMEM16A (ANO1) and TMEM16F (ANO6) in cell migration.

Members of the TMEM16 family have recently been described as Ca(2+)-activated Cl(-) channels. They have been implicated in cancer and appear to be associated with poor patient prognosis. Here, we investigate the role of TMEM16 channels in cell migration, which is largely unknown. We focused on TMEM16A and TMEM16F channels that have the highest expression of TMEM16 channels in Ehrlich Lettre ascites (ELA) cells. Due to the lack of specific pharmacological modulators, we employed a miRNA approach and stably knocked down the expression of TMEM16A and TMEM16F channels, respectively. Migration analysis shows that TMEM16A KD clones are affected in their directional migration, whereas TMEM16F KD clones show a 40 % reduced rate of cell migration. Moreover, TMEM16A KD clones have a smaller projected cell area, and they are rounder than TMEM16F KD clones. The morphological changes are linearly correlated with the directionality of cells. TMEM16A and TMEM16F, thus, have an important function in cell migration-TMEM16A in directional migration, TMEM16F in determination of the speed of migration. We conclude that TMEM16A and TMEM16F channels have a distinct impact on the steering and motor mechanisms of migrating ELA cells.

Deregulation of apoptotic volume decrease and ionic movements in multidrug-resistant tumor cells: role of chloride channels.

Changes in cell volume and ion gradients across the plasma membrane play a pivotal role in the initiation of apoptosis. Here we explore the kinetics of apoptotic volume decrease (AVD) and ion content dynamics in wild-type (WT) and multidrug-resistant (MDR) Ehrlich ascites tumor cells (EATC). In WT EATC, induction of apoptosis with cisplatin (5 muM) leads to three distinctive AVD stages: an early AVD(1) (4-12 h), associated with a 30% cell water loss; a transition stage AVD(T) ( approximately 12 to 32 h), where cell volume is partly recovered; and a secondary AVD(2) (past 32 h), where cell volume was further reduced. AVD(1) and AVD(2) were coupled to net loss of Cl(-), K(+), Na(+), and amino acids (ninhydrin-positive substances), whereas during AVD(T), Na(+) and Cl(-) were accumulated. MDR EATC was resistant to cisplatin, showing increased viability and less caspase 3 activation. Compared with WT EATC, MDR EATC underwent a less pronounced AVD(1,) an augmented AVD(T), and a delay in induction of AVD(2). Changes in AVD were associated with inhibition of Cl(-) loss during AVD(1), augmented NaCl uptake during AVD(T), and a delay of Cl(-) loss during AVD(2). Application of the anion channel inhibitor NS3728 inhibited AVD and completely abolished the differences in AVD, ionic movements, and caspase 3 activation between WT and MDR EATC. Finally, the maximal capacity of volume-regulated anion channel was found to be strongly repressed in MDR EATC. Together, these data suggest that impairment of AVD, primarily via modulation of NaCl movements, contribute to protection against apoptosis in MDR EATC.

Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases.

Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2-VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE proliferation suggesting that iPLA2-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration.

Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants.

The involvement of group VI Ca(2+)-independent PLA(2)s (iPLA(2)-VI) in in vitro ischemia [oxygen and glucose deprivation (OGD)] in mouse C2C12 myotubes was investigated. OGD induced a time-dependent (0-6 h) increase in bromoenol lactone (BEL)-sensitive iPLA(2) activity, which was suppressed by specific short interfering (si)RNA knockdown of iPLA(2)-VIA. OGD was associated with an increase in iPLA(2)-VIA protein levels, whereas mRNA levels were unchanged. The levels of iPLA(2)-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blot analysis identified a mouse iPLA(2)-VIA homolog to catalytically inactive 50-kDa iPLA(2)-VIA-ankyrin variants previously identified in humans. Both the mRNA and protein levels of this approximately 50-kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA(2)-VIA seemed to predominantly reside at the endoplasmatic reticulum, where it accumulated further during OGD. A time-dependent reduction in cell viability during the early OGD period (3 h) was partially prevented by iPLA(2)-VIA knockdown or pharmacological inhibition (10 microM BEL), whereas iPLA(2)-VIA overexpression had no effect on cell viability. Taken together, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA(2)-VIA activity that decreases cell viability. iPLA(2)-VIA activation may be modulated by changes in the levels of active and inactive iPLA(2)-VIA isoforms.

In vitro and in vivo studies of creatine monohydrate supplementation to Duroc and Landrace pigs.

Duroc and Landrace pigs as well as primary myotubes from these breeds were used to investigate mechanisms behind differences in their response to creatine monohydrate (CMH). Pigs were supplemented with 0, 12.5, 25 or 50g CMH/d for 5 days (n=10 per treatment and breed). Plasma levels of creatine increased dose-dependently in both breeds, while muscle-creatine phosphate content increased only in the Duroc pigs. (1)H NMR metabolic profiling showed a tendency towards clustering according to CMH supplementation only among Duroc pigs, revealing a stronger response compared to Landrace pigs. The abundance of insulin-like growth factor I and myostatin mRNA was decreased by CMH supplementation while that of type 1 IGF-receptor and creatine transporter was unaffected. Protein synthesis, increased in the myotubes from both breeds, indicating protein accretion, but no effect was observed on the mRNA abundance of IGF-I, type 1 IGF-receptor, myostatin or the creatine transporter in myotubes.

Activation of PLA2 isoforms by cell swelling and ischaemia/hypoxia.

Phospholipase A2 (PLA2) activity is increased in mammalian cells in response to numerous stimuli such as osmotic challenge, oxidative stress and exposure to allergens. The increased PLA2 activity is seen as an increased release of free, polyunsaturated fatty acids, e.g. arachidonic acid and membrane-bound lysophospholipids. Even though arachidonic acid acts as a second messenger in its own most mammalian cells seem to rely on oxidation of the fatty acid into highly potent second messengers via, e.g. cytochrome P450, the cyclo-oxygenase, or the lipoxygenase systems for downstream signalling. Here, we review data that illustrates that stress-induced PLA2 activity involves various PLA2 subtypes and that the PLA2 in question is determined by the cell type and the physiological stress condition.

Downregulation of taurine uptake in multidrug resistant Ehrlich ascites tumor cells.

In daunorubicin resistant Ehrlich ascites tumor cells (DNR), the initial taurine uptake was reduced by 56% as compared to the parental, drug sensitive Ehrlich cells. Kinetic experiments indicated that taurine uptake in Ehrlich cells occurs via both high- and low-affinity transporters. The maximal rate constant for the initial taurine uptake was reduced by 45% (high-affinity system) and 49% (low affinity system) in the resistant subline whereas the affinity of the transporters to taurine was unchanged. By immunoblotting we identified 3 TauT protein bands in the 50-70 kDa region. A visible reduction in the intensity of the band with the lowest molecular weight was observed in resistant cells. Quantitative RT-PCR indicated a significant reduction in the amount of taurine transporter mRNA in the resistant cells. Drug resistance in DNR Ehrlich cells is associated with overexpression of the mdr1 gene product P-glycoprotein (P-gp). Using 5 progressively DNR resistant Ehrlich cell sublines with different P-gp expression pattern no correlation between taurine uptake and P-gp expression was found. Taurine uptake in MDR1 transfected NIH/3T3 mouse fibroblasts was in contrast to the findings in Ehrlich cells increased compared to the parental fibroblasts. It is concluded that the reduced taurine uptake in resistant Ehrlich cells reflects a down regulation of the taurine transporter at the mRNA and protein level and it is most probably not related to P-gp overexpression.

Sodium pertechnetate scintigraphy in detection of Meckel's diverticulum: is it usable?

OBJECTIVE: To evaluate the results of 99mTc-Na-pertechnetate scintigraphy in children presenting with symptoms suspicious of Meckel's diverticulum (MD). METHOD: Retrospective study. A total of 55 99mTc-Na-pertechnetate scintigraphies in 53 patients were compared with the results from surgery and other diagnostic procedures and available section reports during the period from 1 Jan. 1981 to 1 Jan. 1996. RESULTS: There were four positive scintigraphies. Three patients underwent a laparotomy and an MD was found. The fourth patient was not operated on and no rebleeding was seen after an observation period of 4 years. A total of 51 scintigraphies were negative. In this group two cases with an MD were found at a later laparotomy for other reasons. We found a sensitivity of 60% on 99mTc-Na-pertechnetate scintigraphy in the detection of MD, a specificity and accuracy of 98% and 87%, respectively. CONCLUSION: The 99mTc-Na-pertechnetate scintigraphy has only minor diagnostic value in diagnosing patients suspected of having an MD. Scintigraphic examination should be replaced by diagnostic laparoscopy, which has been reported to be safe and effective and offers the possibility of laparoscopic resection of the MD.

Dose relation in the prevention of proximal vein thrombosis with a low molecular weight heparin (tinzaparin) in elective hip arthroplasty.

This is a review of a double-blind, prospective study comparing the thromboprophylactic efficacy and safety of two different prophylactic regimens of a low molecular weight heparin (tinzaparin) in 250 consecutive patients (aged < or 18) undergoing primary elective hip arthroplasty. Regimen 1: 75 U anti-Xa/kg BW (actual range 63 to 91) once daily started 12 hours before operation; and regimen 2: 50 U anti-Xa/kg BW (actual range 41 to 71) once daily started 2 hours before operation. Both regimens were administered in a weight-adjusted fashion and were continued for 7 days after operation or until full mobilization. Efficacy was evaluated by occurrence of postoperative deep vein thrombosis (DVT) diagnosed by bilateral ascending phlebography on day 7 +/- 2 after operation, and the venograms were evaluated in an assessor blind fashion by a panel of three expert radiologists. Safety was evaluated by the amount of blood lost and transfusion requirements during and after the operation; all bleeding complications, reoperations, adverse events and deaths were observed during the study. A 3-month follow-up on survival and occurrence of thromboembolism was performed on all randomized patients. The result was a significantly better protective effect against proximal DVT by regimen 1 compared with regimen 2. This was achieved with improved safety in terms of a significantly decreased need for blood transfusions during operation and fewer wound complications in the postoperative period in favor of regimen 1. Therefore, tinzaparin administered in a dosage of 75 U anti-Xa/kg BW 12 hours before surgery is significantly more protective against proximal DVT and safer than the standard regimen of 50 U anti-Xa/kg BW started 2 hours before surgery in patients undergoing primary elective hip arthroplasty.

Haemostatic aspects of recombinant human erythropoietin in colorectal surgery.

OBJECTIVE: To find out whether recombinant human erythropoietin (r-HuEPO) given perioperatively has any effect on haemostatic activity in patients undergoing elective colorectal resection. DESIGN: A placebo-controlled double-blind study. SETTING: Odense university hospital, Denmark. SUBJECTS: 24 patients undergoing elective colorectal resection, 13 of whom were given r-HuEPO (Eprex) and 11 placebo. MAIN OUTCOME MEASURES: Concentrations of haemoglobin, tissue-type plasminogen activator and plasminogen activator inhibitor-1; activated partial thromboplastin time; prothrombin time; platelet and reticulocyte counts; blood loss; and transfusions. RESULTS: There was no significant change in fibrinolytic activity, prothrombin time, or activated prothrombin time in the treatment group. Platelet counts differed slightly but not significantly, being higher in the r-HuEPO group. There was a significant increase in reticulocyte counts in the r-HuEPO group. CONCLUSION: R-HuEPO given perioperatively significantly increased erythropoiesis in patients undergoing elective colorectal operations but had no influence on haemostatic activity.

[Surgical treatment of dumping by the Lygidakis' method]. / Operativ behandling af dumping ad modum Lygidakis.

Dumping is a common adverse effect of gastrectomy and severe dumping is one of the most intractable conditions in gastroenterology. When medical treatment and diet are insufficient, different operative techniques have been tried, all with an unsatisfactory rate of success. The Lygidakis technique for operative treatment of postgastrectomy-dumping is presented, and results from operations on four patients presented. The results obtained are promising, and Lygidakis operation may be considered as an important alternative to wellknown operative techniques when medical treatment or diet is without effect on severe dumping.

[Quality development at an emergency unit--an intervention study based on consumer satisfaction]. / Kvalitetsudvikling i skadestuen--et interventionsstudium baseret på brugertilfredshed.

A study among the users of an emergency department was carried out to assess user satisfaction, based upon a questionnaire. The study was split into two periods, before and after a change in procedure in the emergency department, namely that a nurse was to give information about waiting time, make coordinations and fix an order of priority of patients (triage) in the waiting room. Fourteen hundred and twenty-six answers were received, 794 before and 632 after the change. The users questioned after this change of procedure felt that they had been better received, that they had waited for a shorter time and described a better general experience as compared to the users questioned before the change, these differences were significant. There were no difference between the two groups concerning opinions on information about diagnosis, treatment and outcome, whether there was enough time for examination, treatment and information, whether the staff were obliging or whether their expectations had been fulfilled. It is concluded that such changes in procedure in the emergency department are to be recommended.

Perioperative thrombosis prophylaxis with low molecular weight heparins in elective hip surgery. Clinical and economic considerations.

A review and meta analysis of randomized prophylaxis studies in total hip arthroplasty (THA) surgery with the low molecular weight heparin (LMWH) compounds presently marketed in Europe. Thromboprophylaxis with recommended dosages of LMWH was significantly more effective than both placebo (no prophylaxis), dextran 70 and low-dose unfractionated heparin (UH) (5,000 IU thrice daily) in terms of protection against objectively diagnosed deep vein thrombosis (DVT), which is the main source of postoperative pulmonary embolism. The efficacy of LMWH was similar to that of adjusted-dose UH but only 2 studies have been conducted with this regimen so far. When combined with 0.5 mg dihydroergotamine (DHE), UH was as effective as LMWH, but DHE bears a definite risk of circulatory disturbances in the lower limbs. In all studies LMWH prophylaxis was safe under the clinical conditions. A cost-effectiveness analysis based on the reported efficacy and safety of LMWH in the European studies showed that, compared with no prophylaxis, dextran 70, and low-dose UH, LMWH prophylaxis used routinely in patients undergoing THA is more profitable for the health care system due to fewer expenses used on treatment of postoperative thromboembolic complications. LMWH therefore leads to better utilization of the economic resources.

Operative treatment of clavicular nonunion.

Twelve patients treated for clavicular nonunion mainly with plate fixation and bone grafting were reviewed in order to evaluate the treatment. Follow-up was a median of 41 months (20-117). Nine out of 12 patients achieved a good end result, but the primary treatment failed in half of the cases, probably because of short (4-hole) semitubular plates and insufficient postoperative immobilization. We conclude that 4-hole semitubular plates cannot be recommended for treating clavicular nonunion because of a high risk of failure. Sufficient postoperative immobilization should follow plate fixation. If this is respected, plate fixation with bone grafting appears as a good method for treating clavicular nonunion.

Thromboembolic complications after arthroscopy of the knee.

Arthroscopy of the knee has always been associated with a low risk of complications, including thromboembolism. Therefore, few studies have been concerned with the matter. We present two case reports and a review of the available literature concerning thromboembolic complications after arthroscopy.

[Stress fractures]. / Stressfraktur.

The authors present stress fracture (fatigue fracture). Theories about the cause, the underlying mechanism, etiology, pathology, localisation, diagnoses and treatment are discussed, and two case histories are mentioned. Suitable precautions are recommended for runners. The authors would like to emphasize the importance of further investigations including technetium scintigraphic, in cases where stress fracture is suspected.