Taurine demonstrates multiple cellular functions including a central role as a neurotransmitter, as a trophic factor in CNS development, in maintaining the structural integrity of the membrane, in regulating calcium transport and homeostasis, as an osmolyte, as a neuromodulator and as a neuroprotectant. The neurotransmitter properties of taurine are illustrated by its ability to elicit neuronal hyperpolarization, the presence of specific taurine synthesizing enzyme and receptors in the CNS and the presence of a taurine transporter system. Taurine exerts its neuroprotective functions against the glutamate induced excitotoxicity by reducing the glutamate-induced increase of intracellular calcium level, by shifting the ratio of Bcl-2 and Bad ratio in favor of cell survival and by reducing the ER stress. The presence of metabotropic taurine receptors which are negatively coupled to phospholipase C (PLC) signaling pathway through inhibitory G proteins is proposed, and the evidence supporting this notion is also presented. Taurine, 2-amino-ethanesulfonic acid, is one of the most abundant amino acids in mammals . The phyA, GABAB and glycine receptors since the agonists or antagonists of these receptors have little effect on the binding of taurine to taurine receptors i, is maintained at sub-micromolar concentration by calcium sequestering into internal calcium storage pools e.g., mitochondria, endoplasmic reticulum (ER) as well as pumping out to the extracellular space by calcium-ATPase. When neurons are stimulated by glutamate, the [Ca2+]I level is elevated due to influx of calcium from extracellular sources through various calcium channels including NMDA receptors, voltage-gated calcium channels (VGCC) such as L-, N- and P/Q-type, reverse mode of Na+/Ca2+ exchanger as well as release of calcium from the internal calcium storage pools. However, in the presence of taurine, glutamate-induced increase of [Ca2+]i is markedly reduced as shown in Fig It is known that the level of intracellular free calcium, [Ca2+]i is through inhibition of Ca2+ influx via the reverse mode of Na+/Ca2+ exchanger. At the resting membrane potential, Na+/Ca2+ exchanger functions to move Ca2+ out of the cell. However, under depolarizing conditions such as under glutamate stimulation, it reverses its function to facilitate Ca2+ influx [+/Ca2+ exchanger has been suggested to be in part due to in part its membrane stabilizing activity [+/Ca2+ exchanger activity [2+ through the Na+/Ca2+ exchanger and lower tissue Ca2+ content. Taurine also promotes Ca2+ efflux via the Na+/Ca2+ exchanger by increasing [Ca2+]i in the vicinity of the exchanger. i in the absence of extracellular Ca2+ is inhibited by taurine suggesting that taurine inhibits the Glu-induced release of Ca2+ from the internal pools i, and its anti-oxidative stress capacity I by inhibiting calcium influx from various calcium channels including the reverse mode of Na+/Ca2+ exchanger, various voltage-gated calcium channels (VGCC) such as L-, N- and P/Q-type, and glutamate NMDA receptors.1. Taurine reduces glutamate-induced elevation of [Ca2. Taurine inhibits phosphorylation of VGCC resulting in decrease of calcium influx 3. Taurine also reduces the release of calcium from the internal storage pools presumably due to inhibition of phospholipase C.3. Taurine inhibits glutamate-induced activation of calpain and the subsequent hetero-dimerization of Bcl-2 and Bax protein resulting in inhibition of release of cytochrome C and the apoptosis cascade (Fig The authors declare that they have no competing interests.


The mechanical properties of myocardium vary across the transmural aspect of the left ventricular wall. Some of these functional heterogeneities may be related to differences in excitation–contraction coupling characteristics that have been observed in cells isolated from the epicardial, mid-myocardial and endocardial regions of the left ventricle of many species, including canine. Integrative models of coupled myocyte electromechanics are reviewed and used here to investigate sources of heterogeneous electromechanical behaviour in these cells. The simulations (i) illustrate a previously unrecognized role of the transient outward potassium current in mechanical function and (ii) suggest that there may also exist additional heterogeneities affecting crossbridge cycling rates in cells from different transmural regions. Although three-dimensional wall strains are highly inhomogeneous across the transmural aspect of the ventricular wall et al. , 2007. H2+ handling observed in tissue preparations (+ current (Ito1), which is much smaller in endocardial cells (+ channel interacting proteins (2+ channel current (ICaL) density was observed in one study, with endocardial cells showing higher values than epi- and mid-myocardial cells (ICaL density among the three cell types (2+-ATPase (SERCA) is highest in epicardial cells and most probably explains the faster decay of the Ca2+ transient and increased sarcoplasmic reticulum (SR) load observed in these cells . They identified several key distinctions in the time course of unloaded shortening among canine LV epi-, mid- and endocardial cells. Endocardial cells displayed a comparatively longer delay in the onset of shortening, while epicardial cells showed the fastest shortening kinetics among the cell subtypes. Significantly, this study did not show any differences in ICaL density or excitation–contraction (EC) coupling gain among the three myocyte subtypes. Furthermore, the amplitudes of the Ca2+ transient and the peak percentage of cell shortening were not seen to be significantly different among epi-, mid- and endocardial cells. The authors concluded that some, but not all, of the differences in shortening time course were related to AP morphology and Ca2+ handling, leaving room for additional, as yet unidentified, sources of mechanical heterogeneity.2+ handling among cell subtypes.Computational models of ventricular myocyte EC coupling have been useful for investigating the mechanisms of functional differences across the LV wall  is relatively recent, and thus models of the same have developed more slowly than those of myocyte EP  has been modelled in a number of studies .2+ uptake  of 2000 ms was used for all simulations to allow valid comparison with the experiments of The complete electromechanics model was programmed in AP-clamp protocols were simulated by prescribing the membrane voltage with an appropriate time-varying function rather than allowing it to be determined by membrane currents. The epicardial and endocardial AP waveforms used in AP-clamp simulations were digitized from representative traces reported by 2+ handling parameter sets for epi-, mid- and endocardial cells set by 2+ release units from 50 000 to 75 000 to increase the amplitude of the Ca2+ transient at a BCL of 2000 ms to more typically observed levels. This same parameter value has been used by others  in the electronic supplementary material). Ca2+ transients measured experimentally reflect the time course of Ca2+ bound to the indicator molecule ([Ca2+]fluo) and are often calibrated to correspond to the concentration of cytosolic Ca2+. To mimic this measurement, the model reports the time course of [Ca2+]fluo scaled to vary between the minimum and maximum values of [Ca2+]i. We have called this quantity [Ca2+]i/app, the apparent cytosolic Ca2+ concentration. This modification enables direct comparison of model output with experimental measurements.We represented the buffering of intracellular CaIn all simulations where fluo-3 was present, the concentration of this indicator in the cytosol was assumed to be 15 μM, corresponding to the concentration of fluo-3 used in bathing solutions by 2+ contraction dynamics under various conditions. Two Ca2+ input modes were used in these simulations. The first allows myocyte contraction and shortening to be driven with a prescribed Ca2+ transient, which automatically sources as much Ca2+ flux as needed to maintain the command [Ca2+]i signal. This mode was used to study the shortening response to specific Ca2+ transients.The myofilament model of 2+ clamp was created whereby a fixed Ca2+ flux, Jclamp, was used to drive shortening. This approach is similar to the one originally used to estimate Ca2+ flux across the SR . The model displayed unloaded cell shortening characteristics of latency to onset, time to peak and peak percentage of shortening that agreed with mean experimental values to within the reported standard deviations (90) was 222 ms, compared with a mean value of 265 ms  showed a latency to the onset of 16 ms , time to peak of 161 ms (versus 193 ms), RT90 of 489 ms (versus 566 ms) and peak cytosolic Ca2+ concentration of 580 nM (versus 663 nM). The APD of the endocardial model was 326 ms, compared with a mean value of 266 ms reported in experiments at the same BCL of 2000 ms . Peak percentage cell shortening matched measurements within the reported standard deviation for all three cell types (not shown), indicating a reasonable relation between peak systolic [Ca2+]i and myofilament Ca2+ sensitivity.The transmural trend in APD achieved in the original model . The epicardial and mid-myocardial parameter sets both yielded Ca2+ transients with a rapid time to peak that was not fully consistent with measured representative traces or mean values  using AP-clamp conditions. Initial conditions for all model state variables were taken from the mid-myocardial model run to steady state, after which a single beat was elicited in response to a representative endocardial or epicardial AP digitized from the literature (Since the model showed a strong dependence of SR Ca2+ channel currents (ICaL), Ca2+ transients and cell shortening in response to epicardial and endocardial AP waveforms showed large differences in amplitude and time course , corresponding to the greater amount of triggering Ca2+ in the initial phase of ICaL. The endocardial AP-elicited Ca2+ transient reached a peak magnitude above 50 per cent of that of the other transient, despite the fivefold smaller initial trigger current through a longer secondary phase of ICaL . The onset of unloaded shortening was plainly different between the two cases, with shortening in response to the endocardial AP starting approximately 30 ms after that of the epicardial AP (figure 3c). Peak unloaded cell shortening in response to the epicardial AP was reached approximately 100 ms faster than that observed under endocardial AP, and was approximately 30 per cent greater in magnitude.Differences in rdial AP b, corres2+ transient time course and unloaded shortening dynamics, we simulated an unloaded shortening event under a specified or ‘clamped’ Ca2+ transient corresponding to a scaled version of the epicardial AP-elicited transient . The transient was scaled such that its peak corresponded in magnitude with that of the endocardial transient in an attempt to isolate the effects of transient shape from those of absolute magnitude. Unloaded shortening during Ca2+ clamp  displayed shortening latency, time to peak and time from peak to 90 per cent relaxation all well within 10 per cent of those same values in epicardial AP-driven shortening , showing that the time course of the Ca2+ transient was the primary determinant of shortening dynamics on this scale. Surprisingly, the contraction driven by the scaled epicardial Ca2+ transient resulted in a 30 per cent reduction in peak shortening compared with that from the endocardial AP-clamp, in spite of sharing the same absolute magnitude of peak cytosolic Ca2+.To understand better the relationship between Ca2+ transients for mid-myocardial and epicardial cells limited the ability of the electromechanics model to discern potential sources of transmural heterogeneity downstream of Ca2+ handling. We therefore simulated unloaded shortening driven directly by representative Ca2+ transients measured experimentally in the three cell types.Discrepancies between model-predicted and experimentally observed Ca2+ transient and shortening. The concentration of fluo-3 was set to 15 μM, consistent with the concentration used in experiments (2+]i/app with [Ca2+]i from the model containing no fluo-3 (a). Addition of the fluo-3 had negligible impact on the time to peak Ca2+, but lowered the magnitude of the peak by approximately 20 per cent through buffering of free Ca2+. Time from peak to 90 per cent decay of [Ca2+]i/app was increased by 40 per cent over the same value calculated from [Ca2+]i. This prolongation of the Ca2+ transient is caused by competition for free Ca2+ between the high-affinity indicator and the usual Ca2+ uptake pathways, but also possesses an apparent component that reflects the dissociation kinetics of the dye. Overall, this simulation suggests that the apparent Ca2+ transient as assessed by indicator fluorescence is blunted at its peak and decays more slowly in comparison with the actual transient in the absence of this exogenous buffer.Before doing this, we added a simple representation of fluo-3 buffering and kinetics to the electromechanics model  was used to drive shortening in the myofilament model . Peak shortening was reduced by approximately 10 per cent, while the time to peak shortening and RT90 were increased by 32 and 15 per cent over original values, respectively. These differences indicated the need for a method to estimate the true time course of intracellular Ca2+ from fluorescence measurements.To assess the type and magnitude of errors that could be introduced by driving cell shortening directly with measured indicator fluorescence, i/app and [Ca2+]i estimated using flux-clamp (c). These included increased magnitude and faster relaxation for the estimated transient. The estimated shortening in the absence of fluo-3 generated with the flux-clamped model  was compared with shortening driven directly with the fluorescence data  using the same myofilament model parameters in both cases. The contraction driven by fluorescence displayed differences in magnitude of shortening (−15%), time to peak (+7.6%) and RT90 (+28.5%) compared with flux-clamp shortening.We used the flux-clamp protocol to estimate the true Caial cell a can be ux-clamp c. These 2+ relationship possessing a half-activation value (Ca50) and Hill coefficient (nH) of approximately 1.0 μM and 3.5, respectively. Latency to the onset of shortening using these parameters was 12 ms slower than the experimentally reported mean; however, this may be attributed to noise in the original fluorescent signal or error in digitizing the trace, as even small differences in the initial rising phase of the Ca2+ transient have a strong effect on shortening latency. Additionally, RT90 was 54 ms shorter than the experimentally reported mean of 312 ms. This is most probably due to a secondary, slower time constant of relaxation seen in the representative shortening traces for mid- and endocardial cells (cf. figure 1 in figure 5a). The myofilament model parameters adjusted to match the mid-myocardial shortening response also resulted in good reproduction of reported values for endocardial cells (figure 5b). Shortening RT90 for the mid- and endocardial cell responses showed the same systematic deviation from reported mean values and displayed the correct transmural trend (figure 5b). Peak cell shortening under endocardial flux-clamp was within the experimentally reported variation for this value (not shown).The parameters of the myofilament model were adjusted until time to peak shortening and shortening magnitude under mid-myocardial cell flux-clamp were within the standard deviations of measured mean values (90 (b). Time to peak shortening and RT90 were both slower than experimental means by 38 and 101 ms, respectively. These results were strongly suggestive of an underlying difference in myofilament properties between epicardial cells and the other two cell subtypes.Shortening simulated using the base myofilament model parameter set in response to epicardial flux-clamp did not show good agreement with experimental values of time to peak and RT90 b. Time t2+ contraction dynamics between the model and experimental measurements in epicardial cells. As many of the proposed changes affected myofilament Ca2+ sensitivity, the flux-clamp protocol had to be repeated for each one in order to fit a new driving flux. 90 for each hypothesis tested in comparison with the original parameter set (BASE in 2+ sensitivity (SENS), increased myofilament cooperativity (COOP) and a combination of increased titin-based stiffness with steeper cooperative activation (TITIN+COOP). The myofilament model parameter sets used to represent each of these hypotheses are shown in the electronic supplementary material.We considered and quantitatively tested several different hypotheses regarding changes to the myofilaments that might reconcile the discrepancies in Ca BASE in . We test BASE in . Passive BASE in , consist BASE in . We also90 below that shown by the BASE model, but not significantly. Overall, the relaxation of shortening in the model was not sensitive to any of the parameters changed in this process. Time to peak shortening was reduced to some extent by all but the SENS parameter set, which increased time to peak. Time to peak was most sensitive to the V1 parameter set, which brought this value to within the experimentally observed variation .Of all the hypotheses tested in the model, none was able to account completely for the differences between the epicardial cell model and experimentally reported values . The V1 2+ transients does not completely explain differences in shortening, a specific alteration in myofilament protein expression reconciles experimental and modelling results.The present work suggests that much of the mechanical heterogeneity observed by 2+ transient. Early repolarization of the AP was seen as the primary modulator of this aspect of Ca2+ morphology . Cooperative recruitment of force slows the rate of force development at submaximal levels of myofilament activation  the time to peak of the Ca2+ transient. Yet the combined effects of Ito1, increased SR content and increased SR reuptake of Ca2+, manifest in the experimentally measured epicardial Ca2+ transient, appear to be insufficient to account for the observed increase in contraction speed.Flux-clamp simulations suggested that mid- and epicardial cells could not share the same Ca2+ and time to peak shortening, which agreed well with experiments. This change alone was the most effective compared with several alternative hypotheses and suggests a transmural gradient in the expression ratio of V1 to V3 myosin, with V1 expression highest at the epicardium. Although no study of which we are aware has examined transmural gradients of myosin isoform expression in canine, others have been performed in rat  and the Ca2+ transient (Ito1 on contraction seen in the diseased myocardium. However, it is clear that heterogeneous Ito1 expression is present in the normal heart with little effect on the strength of contraction (ICaL in endocardial cells to preserve contractile function. Coupling of the EP and myofilament models allowed us to elucidate this subtle behaviour.Modelling of cardiac EC coupling usually stops short of including contraction. Models and experiments have already examined the relationship between early repolarization (primarily via 2+, while experiments suggest that thin filament activation and crossbridge binding alter TnC buffering capacity (2+, the buffering capacity of the myofilament system may also be viewed as cooperative, undergoing large time- and Ca2+-dependent shifts within a single cardiac cycle. In the coupled electromechanics model, this dynamic buffering shortened APD relative to the original EC coupling model with static TnC buffering by reducing the buffering capacity of the myofilaments during the diastolic interval and allowing more rapid decline of [Ca2+]i via SR uptake and extrusion through other mechanisms. Dynamic buffering of Ca2+ by the myofilaments played an especially important role in the flux-clamp protocol we had designed to estimate the Ca2+ transient after removal of the fluorescent indicator. In the absence of the exogenous buffer, the myofilament buffering capacity adjusted itself in a complex nonlinear manner, resulting in changes to Ca2+ transient morphology.The majority of models of myocyte EC coupling assume a constant affinity of TnC for Ca2+ transient based on the time course of fluorescent indicators. We used the electromechanics model to show that such indicators can cause large errors in Ca2+ transient shape. Thus aspects of the EP/Ca2+ handling model allowed us to make greater quantitative use of the myofilament model. In summary, including tightly coupled models of all three subsystems improved the quality of all models involved.Most models of myofilament force production are tested using a clamped Ca2+ transient time to peak in the Greenstein model (cf. fig. 6b in 2+ transients unlike those reported experimentally when run at a matching cycle length of 2000 ms. This discrepancy did not affect the use of the model in elucidating the modulation of contraction time course by Ito1, but did limit the ability of the model to identify potential heterogeneities of myofilament properties. This shortcoming was effectively overcome via the flux-clamp simulations, which allowed the prediction of shortening directly from experimentally reported Ca2+ transients.The models presented here have several limitations. Each of the published models that form the components of the present model contains limitations of its own (2+ load reported in canine epicardial cells (2+ load in epicardial cells relative to the other types at a cycle length of 1000 ms, which is consistent with experimental results. However, the electromechanics model at slower cycle lengths produced only a moderately elevated SR load, once again indicating an incorrect frequency dependence of Ca2+ handling. The result achieved in the model of 2+ by SERCA present in these cells to be implicitly incorporated into the driving flux during the fitting process.We did not fully explore the effects of increased SERCA and higher SR Ca2+ binding kinetics for fluo-3 were taken from experiments performed in vitro (2+ fluxes would not be strongly affected by withdrawing fluo-3 and that the Ca2+ dependence of myofilament buffering and SERCA activity were adequately described by their respective model representations. This series of assumptions seems reasonable, however, as, through the use of the flux-clamp protocol, a set of myofilament parameters was determined that was able to describe the Ca2+ contraction dynamics of both mid- and endocardial cells. Furthermore, this parameter set displayed a steady-state force–Ca2+ relationship similar to those reported experimentally in isolated canine myocytes (The flux-clamp protocol involves several assumptions. The cytosolic concentration of fluo-3 is assumed to be equal to that reported for the bathing solution, and may not reflect the actual value. Furthermore, Cain vitro  and may 2+ handling and myofilament dynamics to explore potential mechanisms by which myocytes within the same heart modulate their EC coupling characteristics. Our work identifies Ito1 and crossbridge cycling kinetics as primary mechanisms underlying transmural variation of myocyte contractile function.We have used an integrative model of ventricular myocyte function that includes electrophysiology, Ca


Acinetobacter baumannii (MDRAB) is an important nosocomial pathogen usually susceptible to carbapenems; however, growing number of imipenem resistant MDRAB (IR-MDRAB) poses further clinical challenge. The study was designed to identify the risk factors for appearance of IR-MDRAB on patients formerly with imipenem susceptible MDRAB (IS-MDRAB) and the impact on clinical outcomes.Multidrug resistant p = 0.001), and the mortality of case patients was associated with shorter duration of “time at risk”, i.e., faster appearance of IR-MDRAB .A retrospective case control study was carried out for 209 consecutive episodes of IS-MDRAB infection or colonization from August 2001 to March 2005. Forty-nine (23.4%) episodes with succeeding clinical isolates of IR-MDRAB were defined as the cases and 160 (76.6%) with all subsequent clinical isolates of IS-MDRAB were defined as the controls. Quantified antimicrobial selective pressure, “time at risk”, severity of illness, comorbidity, and demographic data were incorporated for multivariate analysis, which revealed imipenem or meropenem as the only significant independent risk factor for the appearance of IR-MDRAB . With selected cases and controls matched to exclude exogenous source of IR-MDRAB, multivariate analysis still identified carbapenem as the only independent risk factor . Case patients had a higher crude mortality rate compared to control patients (57.1% vs. 31.3%, Judicious use of carbapenem with deployment of antibiotics stewardship measures is critical for reducing IR-MDRAB and the associated unfavorable outcome. Acinetobacter baumannii is an increasingly important nosocomial pathogen with resistance to multiple antimicrobial agents A. baumannii (MDRAB) usually retained in vitro susceptibility to carbapenems in vitroThere have been many studies on risk factors for the emergence of MDRAB This case control study was designed to identify the risk factors for the common situation of appearance of IR-MDRAB on patients formerly with imipenem susceptible MDRAB (IS-MDRAB). We compared two groups of patients, both with IS-MDRAB at first. IR-MDRAB appeared later in one group but not in the other. Time at risk, quantified antimicrobial selective pressure, severity of illness, comorbidity and demographic data, were incorporated into multivariate analysis. The impact of appearance of IR-MDRAB on clinical outcomes was evaluated as well. The study was deliberately designed for optimization of several important methodological principles of case control studies for risk factor analysis of antibiotic resistance, including control group selection and adjusting for confoundings such as time at risk, quantified antimicrobial selective pressure, severity of illness and comorbidity The study was approved by the ethics review board of the Chang Gung Memorial Hospital Linkou Medical Center in September 2008. Patient consent was not obtained because data were analyzed anonymously.A. baumannii was by conventional biochemical tests Chang Gung Memorial Hospital Linkou Medical Center is a 3000-bed university-affiliated medical center with 308 ICU beds. Identification of Hospitalized patients with one culture of IS-MDRAB and subsequent cultures of MDRAB from the same body site, sampled during the same hospitalization and at least 7 days apart, were included. Patients with succeeding clinical isolates of IR-MDRAB were defined as the cases. Those with all subsequent clinical isolates of IS-MDRAB were defined as the controls. Two hospitalizations separated by less than 30 days were taken as one hospitalization. Blood was considered as the same body site of any culture site, since it was possibly the invasive complication of any infected site. Pleural effusion was taken as part of the respiratory tract. Patients may be included for more than 1 time if they met the inclusion criteria during different occasions, such as different admissions or different episodes of the same admission.The “time at risk” was defined as the time interval between detection of first IS-MDRAB and detection of first IR-MDRAB for case patients, and the time interval between detection of first and last IS-MDRAB for control patients. It is the period of time at risk for appearance of imipenem resistant strains for each individual patient.A. baumannii growth and comorbidities were gathered by reviewing the medical records. Comorbidities included hepatic dysfunction of a bilirubin concentration over 2.5 mg/dl or liver cirrhosis, renal insufficiency of a creatinine level above 2.0 mg/dl or requirement of dialysis, chronic pulmonary disease, cardiac disease, cerebral vascular accident, diabetes mellitus, immune compromise, hematological or solid organ malignancy, and surgery. Immune compromise was defined as corticosteroid use during the hospitalization , human immunodeficiency virus (HIV) infection or acquired immune deficiency syndrome (AIDS), neutropenia (neutrophil count less than 500 cells/mm3) during the time at risk, use of immunosuppressive agents, including chemotherapeutics, within 30 days prior to A. baumannii growth, and concurrent hematological malignancy. Surgical procedures were those within 30 days prior to A. baumannii growth.Age, sex, site of Length of ICU stay, ventilator dependence and doses and duration of all antibacterial agents, during the period of the “time at risk”, were documented. Severity of illness was quantified by a modified APACHE  II score, recorded within 48 hours before or after the day of first clinical isolate of IS-MDRAB t test or Mann–Whitney test for continuous variables as appropriate. The Kolmogorov-Smirnov test or Shapiro-Wilk test was used to assess normality as appropriate. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. All variables with a p value of <0.05 in univariate analysis were included in a logistic regression model for multivariate analysis. All tests were two-tailed, and a p value of <0.05 was considered significant in multivariate analysis.All statistical analyses were performed using SPSS version 15.0. Tests performed in univariate analysis were chi-square test or Fisher exact tests for categorical variables and Student With review of the microbiology laboratory database, we identified in 206 patients 209 episodes of infection or colonization of IS-MDRAB, which were with subsequent cultures of MDRAB. Appearance of IR-MDRAB occurred in 49 (23.4%) episodes but not in the other 160 (76.6%). The former were defined as the cases and the later were defined as the controls.A. baumannii growth. The “time at risk” ranged from 7 to 134 days, with a mean of 21.9 days. ICU admissions during the “time at risk” were common (60.8%), as well as the use of mechanical ventilation (47.8%). Disease severity varied with a mean APACHE II score of 18.3. Respiratory tract is the predominant site of growth (57.9%). Secondary bacteremia occurred in 16 (7.8%) of the 205 episodes of defined primary site other than primary bacteremia. In-hospital and 30-day mortality rates were 37.3% and 33% respectively.For the 209 episodes, the mean age was 62±20 year old, presenting a population of seniority. Male predominated (66%) and comorbidities were common. About 40% of them received surgical procedures within 30 days prior to p = 0.02). More case patients were with isolates from the respiratory tract  and more control patients were with isolates from the wound . Incidence of bacteremia was comparable for cases and controls . The comorbidities were similar, except that there were more cases with hepatic dysfunction  and more controls with musculoskeletal system and soft tissue surgeries . Case patients were with longer time at risk , greater ICU length of stay , more ventilator days , and higher APACHE II scores .Comparing cases with controls, there was no difference in predominant sex, but the cases were older , imipenem or meropenem (10 p<0.001), and clindamycin . In contrary, control patients were exposed to more first generation cephalosporins  and gentamicin   . Among a= 0.001) .p>0.05). Gender, comorbidities, ICU length of stay, ventilator days, and APACHE II scores were also similar. “Time at risk” was longer for cases than controls . Vancomycin or teicoplanin (7.5 p = 0.03), imipenem or meropenem (10 p<0.001) and ciprofloxacin  were more often used in case patients. Multivariate analysis reached the same conclusion that exposure to carbapenems was the only significant independent risk factor for the appearance of IR-MDRAB   .p = 0.001 and 49.0% vs. 28.1%, p = 0.007). Case patients who survived were more likely with isolates from the wound . MDRAB bacteremia was noted in 6 of the 49 cases, with 5 of them of IR-MDRAB. In-hospital mortality for the 5 patients was 80% (4/5). One patient of chronic ulcer infection survived, even without specific antimicrobial therapy.The case patients had higher in-hospital and 30-day mortality , higher APACH II scores , longer ventilator days , less exposure to oxacillin (p = 0.02), and shorter “time at risk” . However, in-hospital mortality was significantly associated only with the length of “time at risk” in multivariate analysis . The sooner the IR-MDRAB emerged, the less chance the case patients would survive.Associations between in-hospital mortality and various conditions during the period of “time at risk” were analyzed for the 49 cases with appearance of IR-MDRAB. Cases with mortality had more hepatic dysfunction .Combination of carbapenem and sulbactam is one of the recommended regimens for IR-MDRAB eradication, based on the Our study reached the conclusion that the only independent risk factor for appearance of IR-MDRAB in patients formerly with IS-MDRAB is the use of carbapenem. The “time at risk”, the period of time at risk for appearance of IR-MDRAB, is a very important confounding factor to be adjusted because the probability of appearance increases with the length of the time The clinical impact of MDRAB is controversial. Some studies showed prolonged hospital and ICU stays and increased mortality; however, there was no attributable mortality if severity of illness and underlying diseases had been controlled Our samples were clinical, instead of surveillance, isolates. All cultures were requested by clinicians based on clinical discretion. Studies with surveillance isolates may reach different conclusions. However, clinical isolates are more clinically relevant and will provide insightful information for clinical practice. The serial isolates were from the same body sites of each individual patient. It implied possibility of either susceptibility conversion from formerly imipenem susceptible strains to resistance or resistant strain selection. It was also possible that some resistant strains were acquired from an exogenous source. Our effort for this problem was to select cases and controls for a matched control study. The selected cases and controls, matched for age and location and date of hospitalization, were with similar theoretical likelihood of acquiring resistant strain from the environment. Besides, IR-MDRAB on subsequent cultures of less than 7 days were excluded by our arbitrary definition that they were more likely from exogenous sources.Acinetobacter species possessed a wide array of β-lactamases and other enzymes for carbapenem resistance, encoded by transposable elements, plasmids or chromosomes Acinetobacter expanded from undetectable minority to predominant clones as carbapenem had eradicated the majority of sensitive ones. Although our data somehow suggested an inducible mechanism for carbapenem resistant strains to emerge under the selective pressure of carbapenem, there has been so far no microbiological evidence linking any resistance mechanism to such a phenomenon.It is interesting to understand the microbiology behind this clinical phenomenon. Numerous reports suggested causal associations between quantities of antibiotic used and the development of resistance with hospital-wide or nationwide data


Polyadenylated, mRNA-like transcripts with no coding potential are abundant in eukaryotes, but the functions of these long non-coding RNAs (ncRNAs) are enigmatic. In meiosis, Rec12 (Spo11) catalyzes the formation of dsDNA breaks (DSBs) that initiate homologous recombination. Most meiotic recombination is positioned at hotspots, but knowledge of the mechanisms is nebulous. In the fission yeast genome DSBs are located within 194 prominent peaks separated on average by 65-kbp intervals of DNA that are largely free of DSBs.prl class. DSB peaks map to ncRNA loci that may be situated within ORFs, near the boundaries of ORFs and intergenic regions, or most often within intergenic regions. Unconditional statistical tests revealed that this colocalization is non-random and robust (P≤5.5×10−8). Furthermore, we tested and rejected the hypothesis that the ncRNA loci and DSB peaks localize preferentially, but independently, to a third entity on the chromosomes.We compared the genome-wide distribution of DSB peaks to that of polyadenylated ncRNA molecules of the Meiotic DSB hotspots are directed to loci that express polyadenylated ncRNAs. This reveals an unexpected, possibly unitary mechanism for what directs meiotic recombination to hotspots. It also reveals a likely biological function for enigmatic ncRNAs. We propose specific mechanisms by which ncRNA molecules, or some aspect of RNA metabolism associated with ncRNA loci, help to position recombination protein complexes at DSB hotspots within chromosomes. Small (∼20–30 nt) ncRNAs such as siRNA and miRNA can trigger the formation of euchromatin or heterochromatin, affect positively or negatively transcription, induce the deadenylation of mRNA, trigger the targeted degradation of mRNA, and regulate positively or negatively the translation of functional mRNAs  for hotspots across the genome In meiosis, a combination of crossover recombination structures (chiasmata) and sister chromatid cohesion distal to chiasmata help to align homologous chromosome pairs and ensure their proper segregation in the first meiotic division Figure 1i.e., genes) and in intergenic regions (IGRs), but they localize preferentially to large IGRs. Other than this general bias towards IGRs, no discrete features which might be responsible for DSB hotspot clustering were identified The meiotic recombination protein Spo11 (Rec12 in fission yeast) is a conserved, topoisomerase II-like enzyme which introduces dsDNA breaks (DSBs) that initiate recombination rec7 gene encompasses three polyadenylated, transcript from opposite strand RNA molecules, tos1, tos2, and tos3  (prl class of ncRNA molecules and the positions of prominent DSB clusters that initiate meiotic recombination.The 194 prominent DSB peaks encompass about 700 kbp (5.6%) of the genome sequence and those peaks are separated by regions essentially devoid of DSBs prl data set . The complete set of all detectable DSB peaks (weak and prominent) map to about 10.6% of the genome sequence prl molecules to fall within a peak by chance, and have 97.4% confidence that the number falling within a peak would be 12 or less. The probability of observing 24 or more occurrences out of 68 is low (P = 5.5×10−8)  across the genome.The preceding analyses considered the 194 prominent DSB peaks that collectively contain most of the meiotic DSBs catalyzed by Rec12 (Spo11). However, there are an additional 159 “weak” DSB peaks that encompass less than 5% of the genome and whose aggregate area integrals contribute 12% of the total DSBs measured across the genome prl loci are within IGRs prl loci and DSB peaks localizing preferentially, but independently, to IGRs. To explore this possibility further, we restricted the analysis to the 40% of the fission yeast genome comprised of IGRs, and we computed the corresponding conditional probabilities under the binomial distribution and null hypothesis of no association. DSB peaks (prominent and weak) cover 22% of DNA within IGRs, prominent peaks cover 14% of DNA within IGRs, and 59 prl loci are specific to IGRs. Twenty four of the 59 IGR-specific prl loci (41%) occur within a DSB peak (P = 9.6×10−4), 18 of which (31%) occur within a prominent peak (P = 8.6×10−4). We conclude that the intimate association between DSB peaks and prl loci does not arise from independently preferential localization of each to an IGR. Rather, the DSB peaks colocalize specifically with ncRNA loci that may be situated within ORFs, near ORF-IGR boundaries, or most often within IGRs  prl class help to activate DSB hotspots. This process may be conserved, because in mice one ncRNA has been shown to map to a well-defined meiotic recombination hotspot Transcripts of the The vast majority of fission yeast ncRNAs remain undiscovered prl loci are embedded entirely within DSB peaks , then the fraction of prl loci associated with DSB peaks also increases dramatically ? We propose two, not mutually exclusive, hypotheses on mechanism.prl-dependent chromatin remodeling, due either to some aspect of transcription at prl loci or mediated by the ncRNA molecules themselves, creates a preferential site for the nucleation of recombination protein complexes. This hypothesis fits comfortably within the prevailing orthodoxy, which posits that the relative accessibility of DNA in regions of “open” chromatin has a role in hotspot activity  base pair with their targets (RNA or DNA) and thereby deliver protein complexes to those targets e.g., as is implicated for the siRNA-containing RITS complex). Alternatively, it may involve the recognition of R-loop structures by protein complexes . In either case, the base pairing between ncRNA molecules and homologous chromosomal DNA would guide recombination to hotspots.Small ncRNAs . Second, they reveal a likely biological function for many of the enigmatic, polyadenylated ncRNA molecules that are so abundant in eukaryotes.One of our two alternative hypotheses is that the polyadenylated ncRNA molecules help to position meiotic recombination. For context, this hypothesis is consistent with, and might explain mechanistically, some of the many connections between transcription and recombination documented previously see . A few aprl class were identified among 987 random cDNA clones tosprlprl locations annotated in the Agilent microarray spreadsheet (Agilent Technologies). Seventeen prl loci were not annotated in that spreadsheet . These were added manually. One locus (prl52) was annotated twice (on chromosomes I and III). The position for chromosome I was used, as it is congruent with the location listed in the genome database. The distances between each prl locus and its two neighboring DSB peaks (left and right) were determined and tabulated in Excel 2005 . Ends of prl molecules (loci) that map within DSB peaks were assigned distance values of zero. Because 88% of the observed DSBs  were classified previously as falling within prominent DSB peaks Sixty-eight polyadenylated, ncRNA molecules of the P value for the observed number of mappings to DSB peaks was calculated using the exact binomial test of a single proportion. Exact binomial one-sided upper confidence limits were also calculated for the expected number of successes under the null hypothesis. Calculations were performed using the “BINOMDIST” function in a Microsoft Excel spreadsheet . The binomial-proportion estimates, sample sizes, observed success rates, null-hypothesis confidence limits, and calculated P values for each test scenario are given in the main text.Each locus expressing an ncRNA molecule can map either within or outside of a DSB peak. Thus, under random genome-wide placement with constant per-locus “success” probability of mapping within a peak (the null hypothesis), the set of ncRNA loci should approximate the binomial distribution with respect to DSB peaks. We therefore modeled the data using the Binomial distribution. Binomial proportion parameters (success probabilities per locus) were estimated as equal to the fraction of the genome encompassed by “all” and “prominent” DSB peaks. Conditional binomial proportion parameters  were similarly set equal to the fraction of IGRs encompassed by “all” and “prominent” DSB peaks. For every parameter estimate, the


However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.We have measured changes in intracellular Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca The formation of this so-called ‘fertilization envelope’ serves as a slow block against supernumerary sperm entry At the initial stage of fertilization, the interacting egg and sperm become mutually activated. Due to the availability of large quantities of gametes and the easiness of experimental manipulation, this early phase of the fertilization process has been extensively studied in echinoderms. In starfish, it has been clearly demonstrated that the fertilizing sperm undergoes an acrosome reaction in which the sperm head generates a very long tube-like structure called the ‘acrosomal process’ upon contacting the egg jelly coat 2+ in fertilized eggs has made it easier to study the general molecular mechanisms underlying Ca2+ mobilization inside the cell. Studies based on fluorescent Ca2+ indicators and with electrophysiological methods have demonstrated that the release of Ca2+ from intracellular stores is mediated by second messengers such as inositol 1,4,5-trisphosphate (InsP3), cyclic-ADP-ribose (cADPr), and nicotinic acid adenine dinucleotide phosphate (NAADP) 2+ waves 2+ signals in a narrowly defined subplasmalemmal domain 2+ liberation at several discrete spots inside the egg, which then merge to form a broad wave on the cortex which propagates to the center 3 may play a central role in the propagation of this Ca2+ release 3 receptor (InsP3R) with an antibody blocked the fertilization-induced Ca2+ oscillations, suggesting that InsP3 is instrumental in the generation of Ca2+ waves 3 antibodies in echinoderm eggs have not produced the same result 3 receptor, heparin, did not completely block the sperm-induced Ca2+ signals in echinoderm eggs 3 production occurred after the initiation of the Ca2+ wave, raising the possibility that InsP3 might not be responsible for the initial surge of Ca2+2+-releasing mechanism in echinoderm eggs appears to be fundamentally different from that of mammalian eggs with respect to the contribution of InsP3.The massive and rapid release of Ca2+-releasing second messengers have different roles. The rapid and cortex-restricted Ca2+ release by NAADP is reminiscent of the cortical Ca2+ flash induced by the fertilizing sperm 3-evoked Ca2+ wave exhibits a more global mode of propagation. Comparison of Ca2+ signals induced by NAADP and by InsP3 in enucleated postmeiotic eggs has suggested that NAADP initiates the sperm-induced Ca2+ response, while the InsP3 is involved in the later propagation of the Ca2+ wave 3-evoked Ca2+ rise in enucleated eggs displayed comparable amplitude but a slightly delayed kinetics, and failed to trigger cortical granule exocytosis 3-dependent Ca2+ release and the cortical granule exocytosis are under the regulation of unknown mechanisms influenced by nuclear factors such as MPF (Maturation Promoting Factor) In starfish eggs, the different Ca2+ release and cortical granule exocytosis in starfish eggs 2+ signaling and the formation of fertilization envelope, as well as for the control of monospermy and sperm entry.We have recently observed that the actin cytoskeleton plays important roles in modulating intracellular CaA. aranciacus and A. pectinifera) were obtained from the Gulf of Naples, Italy and Mutzu Bay, Japan, respectively. Animals were maintained in circulating seawater at 16°C, and the gonads were dissected from the central dorsal area near the arms and transferred to cold, filter-sterilized seawater (FSW). Fully-grown immature oocytes were isolated as single cells by sieving through gauze several times in cold FSW. Free oocytes were isolated by repeated rinsing and low-speed centrifugation in cold FSW. To obtain postmeiotic eggs, immature oocytes were stimulated with 1-methyladenine (1-MA) as described previously Two species of starfish (3 (Molecular Probes) by microinjection. Caged InsP3 (5 µM pipette concentration) was co-injected with the fluorescent Ca2+ indicator into eggs. To activate the caged InsP3, oocytes were irradiated with 330 nm UV light for 25 sec with the use of a computer-controlled shutter system . Cytosolic Ca2+ changes were detected with a cooled CCD camera  mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20×/0.50 objective. The quantified Ca2+ signal at a given time point was normalized to the baseline fluorescence (F0) following the formula Frel = [F−F0]/F0, where F represents the average fluorescence level of the entire egg. Fluorescent Ca2+ images were analyzed with the MetaMorph Imaging System software . The incubation conditions of jasplakinolide (JAS), latrunculin-A (LAT-A), and heparin are indicated in the figure legends. Unless specified otherwise, the control cells refer to the eggs from the same batches that were treated with the same vehicle for drug delivery.Microinjection of eggs was performed with an air-pressure Transjector (Eppendorf). In this standardized method, the amount of injected material was estimated at 1–2% of the egg volume. Hence, the final concentration of the injected material inside the eggs should have been 50 to 100-fold lower than the concentration in the injection pipette. The fluorescent calcium dye, Calcium Green 488, conjugated to 10 kDa dextran (Molecular Probes) was used in 5 mg/ml pipette concentration with the injection buffer . The same injection buffer was used for delivering heparin (Sigma-Aldrich) and caged InsPF-actin was visualized by two methods, either with or without cell fixation. To visualize F-actin in living cells, starfish eggs were microinjected with 50 µM Alexa Fluor 488-conjugated phalloidin and visualized with confocal microscopy after 10 min incubation. To stain polymerized actin in fixed cells, starfish eggs were treated in 0.5% glutaraldehyde for 1 h and then permeabilized in 1% Triton X-100 (PBS) for 2 h before incubating in 0.1 µM of fluorescent phalloidin  for 2 h. After extensive washing in PBS, eggs were transferred to an experiment chamber and were observed with an Olympus Fluoview 200 laser-scanning microscope with a 60× (1.20 NA) objective. Images of F-actin stained with Alexa Fluor 488-conjugated phalloidin were recorded through a BP 510–540 emission filter. Transmitted light and fluorescent confocal images of the control and the experimental eggs were acquired from the equivalent cytoplasmic planes. For transmission electron microscopy, oocytes were first fixed in 1% glutaraldehyde  for 1 h at room temperature and then rinsed extensively in FSW before being treated 1 h with FSW containing 1% osmium tetroxide. Specimens were dehydrated in increasing concentrations of alcohol and embedded in EPON 812. Sections were stained with 2% uranyl acetate and 0.2% lead citrate and examined with a LEO 912 AB energy filter transmission electron microscope.2+ inside the egg, which leads to the exocytosis of cortical granules and to the subsequent elevation of the vitelline layer 2+ entry, and the formation of the fertilization envelope has not been described in detail. Owing to the large cell size and optical transparency of the cytoplasm, starfish eggs have been an excellent model system for studying intracellular Ca2+ release and morphological changes with the help of fluorescent Ca2+ indicators and imaging systems A. aranciacus egg produced the characteristic cortical Ca2+ flash and the propagation of the Ca2+ wave from the site of sperm-egg interaction. We have noticed that the fertilizing sperm is able to induce Ca2+ signaling and to initiate the vitelline layer elevation while still outside the egg . In addition, the characteristic cortical flash seen in the control egg was abolished in the heparin-treated eggs. The amplitude and kinetics of the intracellular Ca2+ rise were also significantly reduced . Considering that heparin is an inhibitor of InsP3 receptors, the inhibition of Ca2+ propagation was not surprising. The treatment with heparin indeed inhibited the Ca2+-releasing activity of injected InsP3. The average amplitude of the Ca2+ peak seen in control eggs (0.55±0.04 arbitrary units) was strongly reduced (0.25±0.05), but not completely abolished , a well characterized agent that stimulates cortical actin polymerization 2+ waves that represent loci of sperm interaction , while it was 1.6 (n = 10) in the control eggs. Although the fertilization cones in both cases were rich in F-actin , the heperaction . The moseraction . This efd by JAS . On the trol egg . An addi release , as the  release . F-actin release .As mentioned earlier, alteration of the cortical actin cytoskeleton by heparin leads to the polyspermic formation of fertilization cones. To test if heparin also affected sperm entry, the fertilization process was monitored with a CCD camera. Although heparin-treated eggs produced at least 5 times more fertilization cones than control eggs in a given focal plane, the frequency of actual sperm entry through these fertilization cones was significantly lower. Whereas 9 out of 10 fertilization cones in the focal plane of control eggs exhibited successful sperm entry, the success rate in the heparin-treated eggs was 14 out of 28 (n = 8). Similar results were obtained when sperm were prestained with the DNA dye Hoechst 33342 (not shown). The increased occurrence of ‘empty’ fertilization cones in heparin-treated eggs enabled us to monitor the abortive procedure of sperm entry. In the heparin-treated eggs, sperm initially penetrated the jelly coat , but in 3 receptors but also induced actin hyperpolymerization ] was nearly 5 times higher than the dose that would completely block the Ca2+ channel activity of InsP3 receptors in isolated microsomes 3 introduced into the cell, this was two fold lower than the levels of endogenous InsP3 measured under physiological conditions at the height of the Ca2+ liberation peak 2+ signal could have been attributed to the possibly insufficient efficacy of heparin as an InsP3R inhibitor inside a cell as large as the starfish egg. However, it appears more logical to assume that the InsP3 receptors were completely repressed by heparin as they would have been in vitro3 might also liberate Ca2+ inside the egg by a mechanism independent of the InsP3Rs. Alternatively, and perhaps more plausibly, heparin may affect the generation of Ca2+ signals by a mechanism additional to the inhibition of InsP3R. The finding described here showing that heparin substantially inhibited the cADPr-evoked Ca2+ signals Click here for additional data file.Data S2Heparin hyperpolymerizes cortical actin. A. aranciacus oocytes were treated with 1-MA for 1 h to induce meiotic maturation, and then microinjected with either buffer (A), heparin (B), or De-N-sulfated heparin (C). Following cell fixation with glutaraldehyde, F-actin was stained with fluorescent phalloidin. The hyperpolymerization of cortical actin seen in heparin-treated eggs (B) was not evident in the eggs pre-injected with the structural analog of heparin (C).(0.99 MB TIF)Click here for additional data file.Data S3A. aranciacus. The still-shot images of this movie clip were described in detail in The video animation of the failed sperm entry in heparin-treated eggs of (2.81 MB AVI)Click here for additional data file.


Although it has been clearly demonstrated in functional experiments that calmodulin (CaM) binding to intracellular domains of TRP channels is involved in this process, the molecular mechanism remains elusive. In this study, we provide experimental evidence for a comprehensive molecular model that explains Ca2+-dependent facilitation of TRPV4. In the resting state, an intracellular domain from the channel N terminus forms an autoinhibitory complex with a C-terminal domain that includes a high-affinity CaM binding site. CaM binding, secondary to rises in intracellular Ca2+, displaces the N-terminal domain which may then form a homologous interaction with an identical domain from a second subunit. This represents a novel potentiation mechanism that may also be relevant in other Ca2+-permeable channels.Several Ca Often, this provides a negative feedback mechanism that either prevents excessive, potentially deleterious Ca2+ influx into the cell, or defines the time course of channel activity. Indeed, CaM-dependent inhibition is seen in most of the non-selective, Ca2+-permeable cation channels of the TRP group 2+-selective epithelial Ca2+ channels TRPV5 and TRPV6 Ca2+-dependent regulation with channel activity being potentiated and inactivated at Ca2+ concentrations attained during agonist-dependent activation 2+-dependent facilitation has also been described for TRPC5 TRPV4 is highly expressed in kidney and was originally found to be activated by hypotonic solutions, suggesting a role in renal osmoregulation 2+-dependent potentiation process 2+-dependent current potentiation is disruption of an interdomain interaction within TRPV4 resulting from CaM binding to a C-terminal site. We provide experimental evidence for an underlying mechanism in which an N-terminal autoinhibitory domain forms a molecular switch that controls facilitation of TRPV4.In a previous study, we identified a CaM binding domain in the C terminus of TRPV4 as the structural basis of the Ca2+-dependent potentiation of TRPV4 Because CaM interaction with a C-terminal binding site in TRPV4 has been shown to be an essential step in the Ca2+-dependent and half-maximal at 3.2 µM Ca2+ , C-lobe (CaM34) or both lobes (CaM1234) at 100 µM Ca2+  in HEK293 cells expressing YFP (not shown) or TRPV4 alone (control), or TRPV4 together with CaM or CaM mutants (2+-free solution (not shown). A current increase upon addition of Ca2+ was observed in all cells except those expressing YFP alone. In the latter, currents were not significantly changed at +100 mV , but significantly decreased at -100 mV , probably due to a decrease in leakage current. CaM slightly increased the TRPV4 response to Ca2+, but the increase is not statistically significant, indicating that in HEK cells, the CaM concentration is not a limiting factor in TRPV4 potentiation. CaM mutants with a dysfunctional C lobe  had no effect  or the positively charged residues (KRA) in the N-terminal interaction domain . In AlphThe same was found when CFP- and YFP- fusion protein of the full-length N and C-termini, respectively, N1, C1,  were tes2+-containing buffer, and intracellular Ca2+ release after stimulation of the endogenous M3 muscarinic receptor resulted in transient decreases in the FRET- and concomitant increases in the donor channel fluorescences, indicating a decrease in FRET efficiency  units.Tracer peptides were synthesized and coupled to carboxyfluorescein . Protein interaction experiments were performed in buffers containing 50 mM Tris, pH 7.5, 100 mM KCl, 0.1% BSA and calcium at the indicated concentrations. Calcium concentrations below 50 µM were buffered with HEDTA, EGTA or EDTA at a buffer concentration of 10 mM. CaM concentrations were 10 µM or as indicated; the tracer peptide concentration was 100 nM. Fluorescence anisotropy was measured in a 384-well black multititer plate  using a Fusion alpha FP instrument (PerkinElmer) at 1 s reading time per well. Fluorescence polarization (FP) was calculated according to:Single-cell GFP-FRET was measured in a fluorescence imaging system. Excitation of donor (CFP) and acceptor (YFP) fluorescence was at 430 nm and 520 nm, respectively. After baseline recording, the acceptor fluorescence was selectively photobleached by 30 cycles of 5 s expositions at 520 nm while measuring the donor and acceptor fluorescences. Both channels were normalized to the emission intensity immediately before acceptor bleaching. . In nominally Ca2+-free solutions, Ca2+ was omitted. The standard intracellular solution contained 110 mM cesium methanesulfonate, 25 mM CsCl, 2 mM MgCl2, 0.362 mM CaCl2, 1 mM EGTA, and 30 mM HEPES (pH 7.2 with CsOH) with a calculated [Ca2+] of 100 nM.  relations were obtained from voltage ramps from −100 mV to +100 mV. The standard extracellular solution contained 140 mM NaCl, 5 mM CsCl, 2 mM CaClMethods S1Supplementary Methods.(0.04 MB DOC)Click here for additional data file.Figure S12+ (filled squares) or 1 mM EGTA (open squares). The AlphaScreen output signal is shown in arbitrary units .AlphaScreen-based CaM interaction assay. Streptavidin-coated AlphaScreen donor- and anti-Flag-coated acceptor beads were incubated with the indicated concentrations of CaM-biotin and 6 nM C2-Flag (A) or C2-Flag and 10 nM CaM-biotin (B). Data points show mean±SEM of measurements in buffer containing 100 µM Ca(0.07 MB TIF)Click here for additional data file.Figure S22+-dependent binding of C2 to CaM or CaM mutants that are partially Ca2+-binding defective. The Ca2+-dependence of C2-biotin binding to wild type CaM-Flag or mutants that are Ca2+-binding deficient in the N lobe (CaM12), the C lobe (CaM34) or both lobes (CaM1234) was measured in an AlphaScreen assay at the indicated Ca2+ concentrations. Half maximal binding was observed at 8.8±1.0 µM, 10.7±1.0 µM, 27.0±1.1 µM Ca2+ for CaM, CaM12 and CaM34, respectively with a common hill coefficient of 2.0 (n = 3 independent experiments in triplicate). CaM1234 did not show measurable C2 interaction. To demonstrate the Ca2+-independence of the assay system itself, a GST-Flag-biotin fusion protein was incubated with the AlphaScreen beads at 100 µM Ca2+ or 1 mM EGTA (inset). The AlphaScreen readout was independent of the Ca2+ concentration.Ca(0.07 MB TIF)Click here for additional data file.Figure S32+. The peptide dots are shown at the start of the respective 20-mer peptide within the TRPV4 sequence graph. Thus, the underlying peptide sequence actually extends 20 amino acids, corresponding to 10 dots, to the right. The positions of the ankyrin domains, the fragments N2 and N3 and peptides P1, P2 and P3 are indicated. (B) Peptide library of the TRPV4 C terminus, probed with biotinylated CaM or N2 fragment. The positions of the C2-fragment and peptides P4 and P5 are indicated.Identification of CaM binding and interdomain interaction sites in TRPV4. (A) A library of 20-mer peptides overlapping by 18 amino acids and covering the TRPV4 N terminus was spotted on filter paper and probed with biotinylated CaM or C2 at 100 µM Ca(0.31 MBTIF)Click here for additional data file.Figure S450 values are given in CaM binding in the CaM interaction sites in TRPV4. (A–E), In fluorescence polarization experiments, the carboxyfluorescein-labeled peptides P1 (A), P2 (B), P3 (C), P4 (D) and P5 (E) were incubated with CaM, CaM mutants or isolated CaM lobes (see (0.11 MB TIF)Click here for additional data file.Figure S5The N2 fragment forms dimers in gel filtration experiments. (A) N2 results in a monophasic elution profile with an apparent molecular size that corresponds with a dimeric form of the protein. (B) Calibration was done with a commercial protein mixture with the indicated molecular weights.(0.07 MB TIF)Click here for additional data file.Table S1Positions of the TRPV4 fragments used in the study.(0.03 MB DOC)Click here for additional data file.Table S2CaM binding properties of the putative CaM interaction peptides.(0.03 MB DOC)Click here for additional data file.


We implied earlier that α1-AAB might have a mechanistic role and could represent a therapeutic target.Agonistic autoantibodies directed at the α1-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α1-adrenergic receptor antibodies (α1-AB). Patient α1-AAB and rabbit α1-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human α1A-adrenergic receptor were incubated with patient α1-AAB and rabbit α1-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient α1-AAB and rabbit α1-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca2+ in cardiomyocytes and induce mesentery artery segment contraction.To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had α1-AAB and rabbit α1-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α1-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.Patient α However, α1-AR blockade was discontinued in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) because of a putative increased risk for heart failure 1-AR blockade was beneficial in heart failure. α1-AR can contribute to cardiomyocyte hypertrophy. Huang et al demonstrated a protective effect of the α1A-AR-subtype in cardiac myocytes and defined an extracellular-regulated kinase (ERK) signaling pathway that is required for myocyte survival 1-AR gene-deleted mice had increased interstitial fibrosis, increased apoptosis, and failed induction of the fetal hypertrophic gene program after pressure overload, indicating that α1A-AR are required for myocardial adaptation to stress α1-AAB) against the α1-AR 1-AAB in 44% 1-AAB. Zhou et al. immunized rats with a peptide from the α1-AR epitope 1-AR antibodies (α1-AB) and cardiac hypertrophy. However, the link between α1-AB formation and cardiac remodeling remains unclear. We have now performed additional studies to elucidate the pathophysiological relevance of α1-AAB. We show that removal of α1-AAB with immunoadsorption is feasible and lowers blood pressure. We raised α1-AB in rabbit and purified the α1-AB from rabbits and α1-AAB from patients by affinity chromatography. After confirming binding and functional specificity of the isolated rabbit α1-AB and human α1-AAB, we performed gene expression analysis in cardiomyocytes and vascular smooth muscle cells (VSMC). We show that both rabbit α1-AB and human α1-AAB can evoke calcium signaling and contract vessel preparations via protein kinase C alpha (PKC-α) activation and transient extracellular-related kinase (ERK 1/2) phosphorylation. Our data suggest that human α1-AAB can induce signaling pathways involved in hypertension and cardiac remodeling, suggesting a potential clinical relevance of α1-AAB.Others and we have described agonistic autoantibodies  featured α1-AAB as assessed by cardiomyocyte contraction assay. Microalbuminuria was detected in 33%, diastolic dysfunction in 87%, left ventricular (LV) hypertrophy in 85%, and reduced ejection fraction in 13% of α1-AAB positive patients. The patient characteristics are outlined in We analyzed α1-AAB positive patients undergoing immunoadsorption. The treatments were performed daily for 5 days. The cardiomyocyte contraction assay documented gradually decreasing α1-AAB activity during the course of the five immunoadsorptions (1-AAB activity persisted for the 180 day observation period (orptions . At the n period . Blood pn period .1-AAB were directed against the first extracellular loop (eL1) in 24% and against the second extracellular loop (eL2) in 27% of the hypertensive patients  measurements to determine the binding affinity and specificity of purified autoantibody fractions. Sensorgrams, depicted in 1-AR immobilized to the SA-sensorchip. This affinity-purified antibody fraction also showed weak cross-reactivity with the biotinylated peptide corresponding to the eL1 of the α1-AR (We purified αhe α1-AR . However1-AR (rabbit α1-AB) and purified the antibody on the same peptide-containing affinity matrix. In SPR experiments, the rabbit α1-AB produced regular association and dissociation kinetics reaching a signal of ∼2500 RU at a concentration of 80 µg/ml (1-AAB from patients and rabbit α1-AB increased the beating rate of rat cardiomyocytes. This effect was dose-dependent (1-AR antagonist prazosin (1 µM).We next raised a peptide antibody against the second extracellular loop of the α80 µg/ml . Purifieependent . The max1-AAB, we performed gene expression studies. We used Affymetrix microarrays for quantification of mRNA expression in cardiomyocytes and VSMC after treatment with purified α1-AAB isolated from three different patients, rabbit α1-AB, human control IgG, and the α1-AR agonist phenylephrine (PE) for 24 h. We identified two genes with increased expression in the Affymetrix array that we then verified by TaqMan RT-PCR. These genes were PLA2-IIA and Cacna1c, as shown in 1-AR antagonist prazosin while no changes in gene expression were observed after the treatment with the control IgG.To elucidate the molecular pathways induced by α1-AAB from patients and rabbit α1-AB could elicit Ca2+ signals. 2+ transients in response to the addition of the purified patient α1-AAB. There was a fast increase in the 340 nm/380 nm ratio, which peaked at less than 60 seconds and declined within 2 minutes to a plateau value slightly above the control level before the addition of the α1-AAB. Thus, α1-AAB potently induced a rise in intracellular Ca2+ in the targeted neonatal cardiomyocytes. To elucidate whether or not this Ca2+ response could be common to an autoantibody-α1-AR loop 2-type interaction, we used a rabbit α1-AB. As shown in 1-AB also generated an acute positive Ca2+ response in the cardiomyocytes with a similar time course as did the human antibody preparation. These responses were not elicited by a control IgG. As illustrated in 1-AAB negative patient was not able to raise the intracellular Ca2+.We tested whether or not α1-AB both were able to constrict the vessel  cells stably transfected with human α1Α-AR (CHO/α1Α-AR) with α1-AAB resulted in PKC-α activation, which was blocked by the peptide P2, corresponding to the binding site of the α1-AAB -kinase activity in CHO/αrylation . Additioblotting . α1-AAB prazosin . A similptide P2 . Fractio1-AAB und generated α1-AB show similar binding characteristics by surface plasmon resonance sensorgram and functional testing. The antibodies induce a Ca2+ dependent signal transduction cascade in VSMC and cardiomyocytes that are both important to the pathophysiology of hypertension and cardiac remodeling. By gene array studies, we showed that the isolated patient α1-AAB and rabbit α1-AB both upregulate two gene types, namely PLA2-IIA and Cacna1c. In a proof-of-concept, non-controlled trial, we show that removal of α1-AAB by immunoadsorption lowers blood pressure in α1-AAB positive patients with intractable primary hypertension.The important findings in this study are that isolated α1-AR, the β-ARs, the Ang II AT1-R, and the platelet-derived growth factor-alpha receptor, as reviewed elsewhere The entire idea of “agonistic” autoantibodies contributing to cardiovascular disease is controversial 1-AAB could contribute to hypertension 1-AAB a more robust scientific basis. Thus, we verified our candidate by generating α1-AB by immunization. Concomitantly, we subjected patients from our collective who harbored α1-AAB to immunoadsorption. We found in our uncontrolled trial that lowering α1-AAB titers was associated with a lower blood pressure. We did not perform a randomized double blind, crossover trial. Nonetheless, our study meets class I (safety) criteria and suggests that a future class II (efficacy) trial might be warranted. We cannot exclude the possibility that short-term effects of the immunoadsorption, such as circulating blood volume changes during apheresis or other confounders contributed to the reduction in blood pressure. Since the reduction in blood pressure persisted for the observation period of 180 days, we believe that removal of α1-AAB by apheresis was the main cause for the blood pressure reduction. Within the five days immunoadsorption period, the level of α1-AAB decreased gradually. Thereafter, α1-AAB could not be detected after successful immuoadsorption in these patients for the entire observation period of 180 days. A similar state-of-affairs has been described for β1-AAB in dilated cardiomyopathy after immunoadsorption. These data suggest that immunomodulation by removal or neutralization of the AAB might represent a potential therapeutic strategy Earlier, we speculated that α1-AAB and the α1-AB that we generated against the same epitope in rabbits, using the same affinity purification protocol. Since we used the same peptide sequence for epitope mapping in the cardiomyocyte contraction assay, immunization in the rabbit, and isolation in the affinity column, we are confident that we isolated solely α1-AB. Before using this material to learn more about α1-AR signaling in VSMC and cardiomyocytes, we confirmed binding specificity by functional (cardiac contraction assay and ERK 1/2 phosphorylation) and biochemical (surface plasmon resonance measurement) verification methods. We then performed an Affymetrix gene expression study. We followed up on the results to pursue two candidate genes, PLA2-IIA and Cacna1c that were upregulated in the display, as verified by RT-PCR. Both genes contribute to signaling pathways in hypertension and atherosclerosis. Phospholipases A2 are acute-phase reactants and play an important role in digestion and metabolism of phospholipids, as well as in production of precursors for inflammatory reactions. Plasma PLA2-IIA levels are increased in systemic inflammation, including, rheumatoid arthritis and cardiovascular diseases We isolated the detected α1-AAB from patients and the rabbit α1-AB affected intracellular Ca2+ at two different levels, namely the acute, short-term elevation of intracellular Ca2+, and the increased transcript expression of the voltage-gated L-type Ca2+ channel pore subunit. Acute administration of the purified antibodies to neonatal cardiomyocytes produced a typically shaped Ca2+ transient. The onset of the cytosolic Ca2+ response occurred within few seconds reaching its maximum at less than one minute. α1-AR stimulation potentiates L-type Ca2+ current through CaMK II activation in rat ventricular myocytes 1AR and autoantibodies against the AT1-receptor could activate the Ca2+ current 2+ to the antibodies apparently comprises a temporary imbalance of Ca2+ entry through L-type Ca2+ channel and the sarcoplasmic reticulum Ca2+ release on one hand and Ca2+ sequestration into the sarcoplasmic reticulum and Ca2+ extrusion via the Na+/Ca2+ exchanger on the other hand.Another important finding accrued is the fact that both the α2+ current stimulation, we found that long-term activation of the α1-AR pathway by patient α1-AAB and rabbit α1-AB increases transcript levels of the voltage-dependent L-type Ca2+ channel α1C subunit (Cacna1c) in neonatal cardiomyocytes and VSMC. Voltage-dependent L-type Ca2+ channels (L-VDCC) play a crucial role in the regulation of heart rhythm and contractile performance, as well as in smooth muscle tone. The channels open in response to membrane depolarization providing Ca2+ ingress into the cell. In the myocardium, this local rise in Ca2+ concentration triggers massive Ca2+ release from the sarcoplasmic reticulum stores inducing in turn contraction. L-VDCC expressed in cardiac and smooth muscle are multi-subunit proteins containing the α1C, α2/δ, and β subunits. The α1C subunit forms the ion-selective pore, determines voltage sensitivity, and is a binding site for clinically used Ca2+ channel blockers 1-AR stimulation observed herein provides the route for an enhanced Ca2+ ingress which may contribute to Ca2+ imbalances and translate in cardiac dysfunction. Our results are consistent with those of Pignier et al. who reported on increased expression of functional L-type Ca2+ channels and hypertrophy in neonatal cardiomyocytes upon chronic α1-AR stimulation et al. described that in neonatal rat cardiomyocytes activation of the α1-AR pathway was accompanied by attenuated α1C mRNA levels In addition to acute Ca2+ channel pore subunit has been studied intensively 2+ inward current (30–40%) was observed, and the mice developed hypertrophy and severe cardiomyopathy as a function of age. The small sustained ingress of Ca2+ through the channel is capable of elevating PKC-α before the development of hypertrophy. 1-AR-stimulated hypertrophy in cardiomyocytes is mediated via activation of the Ras-Raf-MEK1/2-ERK 1/2 signaling pathway 1-AAB, rabbit α1-AB, or PE. We demonstrated that α1-AAB and α1-AB induced PKC-α stimulation and ERK 1/2 phosphorylation. Finally, we showed that human α1-AAB and the rabbit α1-AB evoke vasoconstriction in a vascular bed.A transgenic mouse with cardiac specific over-expression of solely the Ca1-AAB removal from severely hypertensive human subjects. This trial presents a hypothesis-generating result. We show that isolated α1-AAB from man and rabbits cause gene upregulation important to hypertension-relevant pathways and then document the induction of Ca2+-dependent pathways. We provide evidence that α1-AAB are of potential pathophysiological relevance and could represent a factor contributing to the development of refractory hypertension. Removal or neutralization of α1-AAB might represent a possible therapeutic option. Accordingly, we suggest that the entire gamut of autoimmunity in chronic cardiovascular disease warrants further critical inspection.Taken together, we present data from a limited, uncontrolled, human trail of α1-AAB were asked to undergo five immunoadsorption treatments with an especially prepared column according to procedures described elsewhere Patients requiring three or more medication classes to achieve goal control blood pressure values were recruited from the Franz-Volhard Clinic outpatient department. All had been studied intensively and secondary causes of hypertension had been ruled out. Home and clinic blood pressures in these patients were determined with an automated oscillometric device under standardized conditions. The internal review board approved the study and written informed consent was obtained from all participants. Five patients harboring α1Α-AR were each added in excess (0.05 to 0.1 µg) to the immunoglobulin fraction. The mixtures were shaken and placed in a refrigerator for 1 h. The 100 µl samples were then added to neonatal rat heart muscle cells cultured in 2 ml of medium to a final dilution of 1∶40. The beating rate was counted for 15 sec, 5 and 60 min after the addition of the peptide/immunoglobulin mixture. We tested α1-AAB activity in a neonatal cardiomyocyte assay without and with peptide inhibition. Pulsation rate was exhibited 1 h or 24 h, respectively after incubation with α1-AAB and compared with the spontaneous basal pulsation rate. Eight synchronously contracting cell clusters per flask were counted for 15 sec on a heat table stage.We isolated immunoglobulin fractions from serum samples as described earlier 1-AR (PAPEDETICQINEE) by BioGenes GmbH . The corresponding peptide of the second extracellular loop of the α1-AR was covalently bound to ε-aminocapryl agarose  to yield epitope-specific affinity beads. For coupling the agarose gel matrix (∼1 ml of packed gel) was activated by glutaraldehyde  for 20 min at room temperature on a rotating wheel. The activated gel was extensively washed with PBS and allowed to react with the peptide solution for 3 h at room temperature or overnight at 4°C on a rotating wheel. The coupling reaction was terminated by incubation with 200 mM Tris/Glycine buffer pH 7.2. The affinity beads were poured into a column  and stored with 0.02% sodium acid in buffer A consisting of 50 mM Tris/HCl, 0.5 M NaCl, pH 7.4, at 4°C.We immunized rabbits against a peptide corresponding to the amino acid sequence of the second extracellular loop of the isoform A of human αThe affinity beads were washed with buffer A to remove the sodium acid. Serum samples (5–15 ml) from patients with refractory hypertension or from immunized rabbits were incubated with the affinity beads overnight at 4°C on a rotating wheel. The beads were allowed to settle and the sera were removed and stored for a second round of affinity purification at a given matrix. The beads were washed with buffer A until baseline levels of protein are detected at 280 nm. The antibodies were eluted at room temperature in 1 ml fractions with 50 mM Tris/Glycine, 0.5 M NaCl, pH 2.5. Antibody fractions were immediately neutralized with 0.5 ml of 0.5 M Tris/HCl, 0.5 M NaCl, pH 7.4. The antibody concentration was calculated by measurement the absorption at 280 nm.1A-AR were immobilized at binding levels of 100 relative units (RU) each on parallel lanes of a SA-biosensor chip. Affinity-purified antibodies from patient blood samples were injected in the flow cells at a rate of 20 µl/min in HBSE running buffer consisting of 10 mM HEPES, pH 7.4, 150 mM NaCl, and 3 mM EDTA. The binding was regenerated between binding measurements using 5 mM Tris/Glycine, 50 mM NaCl, pH 2.5, with no decrease in extent measurements over the duration of an experiment. Data were analyzed using the using the BIAevaluation 3.2 RC 1 program. The analysis software corrects for baseline drift during measurements. The curves were fitted to a single-site interaction model. Kd values were calculated by using the formula Kd = koff /kon in which koff and kon is the rate constant of dissociation and association kinetics, respectively.Binding experiments were performed in a BIAcore 2000 Instrument  at 25°C. N-terminally biotinylated peptides corresponding to the first and second extracellular loop of the α1Α-AR (CHO/α1Α-AR) using a pSW104 vector and were cultured in F12 HAM medium supplemented with glutamine, 10% FCS and 1% penicillin/streptomycin as described earlier 1-AAB from different patients (2.5 µg/ml medium), rabbit α1-AB (2.5 µg/ml medium), and with the α1-AR agonist PE  for 24 h in DMEM medium containing 1% serum. Experiments were repeated with three different cardiomyocytes and VSMC preparations. For investigation of protein phosphorylation, cardiomyocytes and CHO/α1Α-AR cells were maintained in serum-free media for 24 h or 4 h, respectively and treated with PE, human α1-AAB or rabbit α1-AB for 5 and 15 min, respectively. For inhibition experiments, prazosin  was added. Five µg of the peptides P2 (APEDET) or P5 (GYVLFS) were given to 2.5 µg of α1-AAB 1 h before cell treatment. For the inhibition of ERK 1/2 activation, CHO/α1Α-AR cells were pre-incubated with PI3-kinase inhibitor LY294002 for 10 min.Rat neonatal cardiomyocytes were prepared from ventricles of 1–2 day-old Wistar rats using a modified method 1-AAB, rabbit α1-AB or PE using the RNeasy Purification Kit . RNA was treated by deoxyribonuclease I (Qiagen). Two µg RNA of cells were transcribed in cRNA with One-Cycle Target labeling and Control Reagents . Non-pooled microarray experiments were performed with cRNA prepared from independent cardiomyocyte cell preparations using Rat Genome 230 2.0 Arrays . After passing the quality control for each experiment a set of RMA normalized expression values have been produced. The log scale robust multi-array analysis (RMA) estimates are based upon a robust average of log2 (B (PM)), where B (PM) are background corrected perfect match intensities We extracted total RNA from cardiomyocytes treated with human control IgG, α18 primer. Real-time PCR experiments were done using the Mx3000P® real-time PCR system  and the Brillant QPCR master mix (Stratagene Europe). Real-time PCR was performed with non-pooled samples from cardiomyocytes and VSMC respectively. Beta-2-microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as endogenous references to normalize expression of a target gene. For every sample three independent runs in triplicates and the relative changes in gene expression were quantified by comparative Ct method cDNA was synthesized from 2 µg of total RNA isolated from cardiomyocytes and VSMC, respectively using PowerScript Reverse Transcriptase  and an Oligo (dT)We described the techniques for confocal microscopy and immunocytochemistry as referenced previously 1Α-AR cells were scraped in lysis buffer  and centrifuged at 10,000 rpm for 10 min. Protein concentrations were measured with Bradford method and equal amounts of proteins were analyzed by Western blotting. ERK phosphorylation was detected by pERK 1/2 antibody . As translational control an ERK antibody  and eukaryotic initiation factor 4E (elF4E) antibody  were used. HRP-conjugated goat anti-rabbit secondary antibody  was used. Detection was performed with the ECL™-substrate  according to the manufactureŕs instructions.Cardiomyocytes and CHO/α6 cells per chamber. After cultivation for four days the medium was removed and cells were washed twice with 10 mM Hepes-buffered Hank's salt solution, pH 7.4 (HBSS). Cardiomyocytes were incubated on HBSS for 60 minutes at 37°C. Then the solution was replaced by 0.5 ml HBSS containing 2.5 µM final concentration of Fura 2-AM  and left for loading in the dark at room temperature. After 30 min the loading solution was aspirated, the cells washed and kept on 0.5 ml HBSS in the dark and at room for another 30 minutes before use. Measurements of cytosolic Ca2+ transients were performed on an IonOptix Fluorescence and Contractility System  equipped with a Leica microscope with a heatable stage. All measurements were carried out at 37°C. Cardiomyocytes were electrically stimulated at 1 Hz and the ratio of 340 nm/380 nm was recorded. After stabilization of the Fura signal the control trace was taken. Antibody preparations were applied in a constant volume of 100 µl of pre-warmed HBSS to give a final concentration of 2 µg/ml. For evaluation of Ca2+ changes peak values were derived from the trace recordings representing the maximum cytosolic Ca2+ achieved in the contracted cell.Neonatal cardiomyocytes were plated onto Labtek four chamber slides  suited for fluorescence measurements at a densitiy of 0.2×101-AAB (5 µg in 5 ml PBS buffer), rabbit α1-AB (50 µg in 5 ml PBS buffer), rabbit IgG  and human control IgG endobulin (50 µg in 5 ml PBS buffer).Vessel rings from superior mesenteric arteries of male Sprague-Dawley rats  were prepared and intracellular membrane potential was measured as earlier described We relied on student's t-tests (adjusted as necessary) for normally distributed data and performed analysis of variance (repeated measures were indicated). P<0.05 was accepted as significant. Data are expressed as mean±SD.