Response of an Escherichia coli-Bound Fluorescent Probe to Colicin E1

The fluorescent probe, 8-anilino-1-napthalenesulfonate (ANS) binds to Escherichia coli, showing an enhanced fluorescence. The interaction of colicin E1 with sensitive cells causes an increase of about 100% in the fluorescence of the bound ANS, and this change at equilibrium has an apparent "all-or-none" nature as a function of E1 multiplicity. Approximately 6 to 8% of the ANS is bound to the cells at equilibrium. The colicin E1-induced fluorescence increase can be attributed partly to an increase in ANS binding and partly to an increase in the fluorescence yield of the bound ANS. The kinetics of the E1-induced fluorescence increase in sensitive cells are very similar to those of the adenosine triphosphate decrease. The phosphorylation uncoupler p-trifluoromethoxy-carbonylcyanidephenylhydrazone also causes a large change in the fluorescence of bound ANS. Colicin E2 or E3 does not cause any fluorescence change, nor does colicin E1 cause fluorescence change with a colicinogenic strain. ANS appears to be a probe of structural or conformational change in the cell envelope that is closely associated with the colicin E1-induced adenosine triphosphate decrease.     

Temperature dependence of synaptic responses in guinea pig hippocampal CA1 neurons in vitro

1. Temperature-dependent properties of synaptic transmission were studied by recording orthodromic responses of the population spike and excitatory postsynaptic potential in CA1 pyramidal neurons of guinea pig hippocampal slices.2. Increasing the temperature of the perfusing medium from 30 to 43°C resulted in a decrease in the amplitude of the population spike (A-PS) and a reduced slope of the excitatory postsynaptic potential (S-EPSP). Bath application of the -aminobutyric acid receptor antagonist, picrotoxin, or a change in the calcium concentration of the perfusate did not affect the A-PS during heating.3. Increasing the strength of the synaptic input to that eliciting a PS with an amplitude 50, 75, or 100% of maximal at 30°C resulted in a significant increase in the A-PS during the middle phase of hyperthermia (35–39°C).4. The long-term potentiation (LTP) induced at either 30 or 37°C showed the same percentage increase in both the amplitude of the population spike and the S-EPSP after delivery of a tetanus (100 Hz, 100 pulses) to CA1 synapses.5. The results of the present study, therefore, indicate that the decrease in CA1 field potential was linearly related to the temperature of the slice preparation, while LTP was induced in these responses during heating from 30 to 37°C.     

Frequent Mutation of rs13281615 and Its Association with PVT1 Expression and Cell Proliferation in Breast Cancer

The q24 band of chromosome 8 （8q24） is frequently amplified in human cancers including breast cancer, and several SNPs （single nucleotide polymorphisms） at 8q24, including rs13281615, have been identified for their association with cancer risks. These SNPs are in a ＂gene desert＂ region, and their functions in cancer development remain to be illustrated, although several of the SNPs appear to influence the genes in the ＂desert＂ in a long-range manner, including the v-myc avian myelocytomatosis viral oncogene homolog （MYC） and the non- protein coding plasmacytoma variant translocation 1 （PVT1）, both of which have been implicated in human cancers. In the current study, we examined rs13281615 for its potential role in breast cancer using normal and cancer tissues from 121 Chinese women with breast cancer. In addition to confirming the association of the GG genotype of rs 13281615 with breast cancer risk, we found that germline GG genotype was significantly associated with estrogen receptor （ER） positivity, higher tumor grade and higher proliferation index. We also found frequent somatic mutations （22/121 or 18.2%） of this SNP in breast cancer. Interestingly, the majority of the mutations （17/22 or 77%） involved a G→ A change, resulting in a decrease in the number of cancers with the GG risk genotype and subsequent loss of GG association with higher tumor grade and proliferation index in cancers. Furthermore, PVT1 expression was increased in cancers, and the increase was associated with the GG genotype of rs13281615. These results suggest that the GG genotype of SNP rs13281615 plays a role in breast cancer likely by influencing PVT1 expression, and that during oncogenesis, ＂protective＂ mutations could occur.     

Evidence for regulation in vivo of the ATP synthase in intact cells of the photosynthetic bacterium Rhodopseudomonas capsulata

(1) The cytoplasmic membrane potential (Δψ) of intact cells of Rhodopseudomonas capsulata, measured either from the uptake of butyltriphenylphosphonium cation or from the electrochromic carotenoid band shift, increased upon illumination (negative on the cytoplasmic side) and then, within the next 20 s, partly declined while the light was still on. In the presence of the F₀ inhibitor venturicidin the light-induced Δψ was increased by 30% and the partial decline was abolished. (2) From the ionic current/Δψ curves for the bacterial membranes it was concluded that the slow, partial decline of Δψ after the onset of illumination was the result of an increase in membrane conductance. The conductance increase seen in the ionic current/Δψ curves was blocked by venturicidin suggesting that it was caused by increased proton flux through the ATP synthase. (3) Analysis of the light-induced changes in adenine nucleotide levels in intact bacterial cells showed that the apparent increase in ATP synthase activity was not the result of a decrease in phosphorylation potential. The data were consistent with either an increase in the catalytic activity of the ATP synthase or with an increase in H⁺ flux through the enzyme without a proportionate increase in the rate of phosphorylation (increased ‘slip’). (4) This slow change in the properties of the ATP synthase, as judged by the venturicidin-sensitive partial decline of Δψ, required a minimum initial value of Δψ. When Δψ was reduced, either by decreasing the actinic light intensity or by adding carbonylcyanide trifluoromethoxyphenylhydrazone the partial decline in Δψ was abolished. (5) The slow change in ATP synthase properties reversed upon darkening the bacterial cell suspension. A second illumination period shortly after the first elicited a smaller initial Δψ and a smaller Δψ decline. The relaxation of the ATP synthase in the dark was measured from the dependence of the initial increase in Δψ after the second illumination period upon the dark-time between the two illumination periods.     

The Effects of Angiotensin Converting Enzyme Inhibitors (ACE-I) on Human N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) Levels: A Systematic Review and Meta-Analysis

Background

Tuberculous pericardial effusion is a pro-fibrotic condition that is complicated by constrictive pericarditis in 4% to 8% of cases. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a ubiquitous tetrapeptide with anti-fibrotic properties that is low in tuberculous pericardial effusion, thus providing a potential mechanism for the heightened fibrotic state. Angiotensin-converting enzyme inhibitors (ACE-I), which increase Ac-SDKP levels with anti-fibrotic effects in animal models, are candidate drugs for preventing constrictive pericarditis if they can be shown to have similar effects on Ac-SDKP and fibrosis in human tissues.

Objective

To systematically review the effects of ACE-Is on Ac-SDKP levels in human tissues.

Methods

We searched five electronic databases (1996 to 2014) and conference abstracts with no language restrictions. Two reviewers independently selected studies, extracted data and assessed methodological quality. The protocol was registered in PROSPERO.

Results

Four studies with a total of 206 participants met the inclusion criteria. Three studies (106 participants) assessed the change in plasma levels of Ac-SDKP following ACE-I administration in healthy humans. The administration of an ACE-I was associated with an increase in Ac-SDKP levels (mean difference (MD) 5.07 pmol/ml (95% confidence intervals (CI) 0.64 pmol/ml to 9.51 pmol/ml)). Two studies with 100 participants further assessed the change in Ac-SDKP level in humans with renal failure using ACE-I. The administration of an ACE-I was associated with a significant increase in Ac-SDKP levels (MD 8.94 pmol/ml; 95% CI 2.55 to 15.33; I² = 44%).

Conclusion

ACE-I increased Ac-SDKP levels in human plasma. These findings provide the rationale for testing the impact of ACE-I on Ac-SDKP levels and fibrosis in tuberculous pericarditis.     

Tumor necrosis factor inhibits K+ current expression in cultured oligodendrocytes

Summary The effects of tumor necrosis factor- (TNF-), a cytokine secreted by activated macrophages, on the electrical membrane properties of cultured adult ovine oligodendrocytes (OLGs) were investigated using the whole-cell voltage-clamp technique. Treatment with recombinant human TNF- (rhTNF) for 24 to 72 hr produces (i) process retraction in some but not all OLGs, (ii) a reduction in the resting membrane potential with no significant change in membrane capacitance or input resistance over control cells and (iii) a decrease in the expression of both the inwardly rectifying and outward K⁺ current. The magnitude of the membrane potential change as well as K⁺ current inhibition was larger in cells with retracted processes. The electrophysiological effects of rhTNF were attenuated when rhTNF was neutralized with a polyclonal anti-rhTNF antibody. The binding of rhTNF to its receptor has been reported to increase GTP binding, to increase GTPase activity of a pertussis-sensitive G protein, and to produce an elevation in intracellular cAMP in other cell types. However, pretreatment of OLGs with activated pertussis toxin failed to attenuate or mimic the effects of rhTNF. Chronic exposure of OLGs to the membrane permeant analogue of cAMP, 8-bromo-cAMP, resulted primarily in an inhibition of the inwardly rectifying K⁺ current, an effect which was less than that produced by rhTNF alone and without any of the associated rhTNF-induced morphological changes. This indicates that the effects of rhTNF cannot be entirely accounted for by an elevation in intracellular cAMP. Cycloheximide (CHX), an inhibitor of protein synthesis, mimicked the effects of rhTNF; however, the effects of rhTNF and CHX were not additive. The finding that both ionic current expression and membrane potential were reduced in cells treated with rhTNF that appeared morphologically normal suggests that abnormal ion channel expression in OLGs precedes and may contribute to eventual myelin swelling and damage.     

Potential changes in the distributions of latitudinally restricted Australian butterfly species in response to climate change

This study assessed potential changes in the distributions of Australian butterfly species in response to global warming. The bioclimatic program, BIOCLIM, was used to determine the current climatic ranges of 77 butterfly species restricted to Australia. We found that the majority of these species had fairly wide climatic ranges in comparison to other taxa, with only 8% of butterfly species having a mean annual temperature range spanning less than 3 °C. The potential changes in the distributions of 24 butterfly species under four climate change scenarios for 2050 were also modelled using BIOCLIM. Results suggested that even species with currently wide climatic ranges may still be vulnerable to climate change; under a very conservative climate change scenario (with a temperature increase of 0.8–1.4 °C by 2050) 88% of species distributions decreased, and 54% of species distributions decreased by at least 20%. Under an extreme scenario (temperature increase of 2.1–3.9 °C by 2050) 92% of species distributions decreased, and 83% of species distributions decreased by at least 50%. Furthermore, the proportion of the current range that was contained within the predicted range decreased from an average of 63% under a very conservative scenario to less than 22% under the most extreme scenario. By assessing the climatic ranges that species are currently exposed to, the extent of potential changes in distributions in response to climate change and details of their life histories, we identified species whose characteristics may make them particularly vulnerable to climate change in the future.     

Six sessions of sprint interval training increases muscle oxidative potential and cycle endurance capacity in humans.

Parra et al. (Acta Physiol. Scand 169: 157-165, 2000) showed that 2 wk of daily sprint interval training (SIT) increased citrate synthase (CS) maximal activity but did not change "anaerobic" work capacity, possibly because of chronic fatigue induced by daily training. The effect of fewer SIT sessions on muscle oxidative potential is unknown, and aside from changes in peak oxygen uptake (Vo(2 peak)), no study has examined the effect of SIT on "aerobic" exercise capacity. We tested the hypothesis that six sessions of SIT, performed over 2 wk with 1-2 days rest between sessions to promote recovery, would increase CS maximal activity and endurance capacity during cycling at approximately 80% Vo(2 peak). Eight recreationally active subjects [age = 22 +/- 1 yr; Vo(2 peak) = 45 +/- 3 ml.kg(-1).min(-1) (mean +/- SE)] were studied before and 3 days after SIT. Each training session consisted of four to seven "all-out" 30-s Wingate tests with 4 min of recovery. After SIT, CS maximal activity increased by 38% (5.5 +/- 1.0 vs. 4.0 +/- 0.7 mmol.kg protein(-1).h(-1)) and resting muscle glycogen content increased by 26% (614 +/- 39 vs. 489 +/- 57 mmol/kg dry wt) (both P < 0.05). Most strikingly, cycle endurance capacity increased by 100% after SIT (51 +/- 11 vs. 26 +/- 5 min; P < 0.05), despite no change in Vo(2 peak). The coefficient of variation for the cycle test was 12.0%, and a control group (n = 8) showed no change in performance when tested approximately 2 wk apart without SIT. We conclude that short sprint interval training (approximately 15 min of intense exercise over 2 wk) increased muscle oxidative potential and doubled endurance capacity during intense aerobic cycling in recreationally active individuals.     

Energetics of Isolated Hepatocyte Swelling Induced by Sodium Co-transported Amino Acids

This study was designed to investigate the energetics of isolated rat hepatocyte swelling due to sodium-cotransported amino acid accumulation in a medium containing either glucose or octanoate as basal substrate. We show that the size of the increase in cytosolic volume is directly correlated with the total amino acid accumulation, which depends on the difference of electrical potential across the plasma membrane. Such a change in cell volume, with either glucose or octanoate, does not modify the mitochondrial volume. Addition of sodium-cotransported amino acids for which the metabolism was avoided showed that the rise in cell volume, per se, did not change the respiratory rate, p, or phosphate potential in either mitochondrial or cytosolic compartments. Conversely, the large increase in oxidative phosphorylation flux was due to the metabolism of amino acids as a consequence of a rise in electron supply for the respiratory chain rather than an increase in cellular ATP demand, as indicated by the increase in cytosolic phosphate potential. Moreover, although we confirm that octanoate addition largely increases the respiratory rate by a process different from uncoupling, we observed that the same overall thermodynamic driving force through the respiratory chain and the same mitochondrial or cytosolic phosphate potential were maintained for much higher oxygen consumption when octanoate was present. We propose that these octanoate effects are due to a decrease in the actual protons/2 electrons stoichiometry as a consequence of a shift in electron supply toward a two-coupling site instead of a three-coupling site. The change in the FADH₂/NADH formation flux ratio in either fatty acid or carbohydrate oxidation explains such results.     

Effect of 2,6-Diisopropylphenol and Halogenated Anesthetics on Tetraphenylphosphonium Uptake by Rat Brain Synaptosomes: Determination of Membrane Potential

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP⁺) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202 ± 8 mV (mean ± SD) while the synaptosomal membrane showed a potential of 78 ± 8 mV (mean ± SD). When anesthetic concentrations (0.6–1 mM propofol, 10–40 M enflurane, 30–50 M isoflurane, 8–15 M halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP⁺ taken up was observed as the anesthetic concentration was increased.     

Does whole body autoregulation mediate the hemodynamic responses to increased dietary salt in rats with clamped ANG II?

The present study was conducted to test the hypothesis that salt-dependent hypertension, in rats with an unresponsive renin-angiotensin system, is characterized by a "whole body autoregulation" hemodynamic profile. To test this hypothesis, rats were chronically instrumented to continuously measure cardiac output (CO) and arterial pressure (AP). A venous catheter was implanted for infusion of saline vehicle (Veh; n = 8) or treatment [enalapril (2 mg.kg-1.day-1) plus ANG II: ANG-NORM (5 ng.kg-1.min-1 ANG II, n = 8) or ANG-HI (10 ng.kg-1.min-1 ANG II, n = 9)] to pharmacologically clamp plasma ANG II. After a 10-day recovery period on a 0.1% NaCl diet, AP and CO were measured continuously for 5 days of control (0.1% NaCl), 7 days of high salt (4.0% NaCl), and 5 days of recovery (0.1% NaCl). Hemodynamics did not change in the Veh group at any time. AP increased by approximately 20 mmHg in the ANG-NORM and ANG-HI groups when NaCl was increased. Hypertension was mediated by an increase in CO of approximately 12% at steady state, with no change in total peripheral resistance (TPR) during the high salt period. AP returned to control levels when dietary sodium was decreased, mediated by a approximately 10% decrease in TPR, with CO remaining elevated. There was no difference in the hemodynamic responses to increased salt between the ANG-HI and ANG-NORM groups. We conclude that the whole body autoregulation hypothesis does not explain the hemodynamic profile of salt-dependent hypertension in rats with an unresponsive renin-angiotensin system.     

SOS-independent mutagenesis in lacZ induced by methylene blue plus visible light

Summary In vitro photosensitization by visible light in the presence of methylene blue (MB-light) produces lesions in M13mpl8 lacZ phage DNA, the lethal and mutagenic potential of which was analyzed after transfection into various bacterial hosts. Mutagenesis was determined with a forward mutation assay using the lacZ gene of M13mp18 as a target. When, MB-light-treated double-stranded (ds) M13mp18 DNA was used to transfect wild-type cells which were not induced for SOS functions, a fivefold increase in mutation frequency was observed at 10% survival compared to that observed with untreated DNA. Mutation frequency obtained with MB-light-treated ds M13mp18 DNA was greater when transfected into the uvrA fpg-1 double mutant than that seen in uvrA, fpg-1, or umuC single mutants or in the wild-type. Sequence analysis shows that in the wild-type strain, MB-light treatment of ds M13mp18 DNA results mostly in single base substitutions. The most frequent base change is the GCTA transversion. MB-light treatment of single-stranded (ss) M13mp18 DNA also results in an increased mutation frequency after transfection into the wild-type strain, yielding mostly GT transversions. Our results show that MB-light-induced mutagenesis is at least partially independent of the induction of SOS functions in Escherichia coli. The mutation spectra suggest that 8-oxo-7,8-dihydroguanine is the major promutagenic lesion in DNA.     

中国东北落叶松属3种植物潜在分布对气候变化的敏感性分析

该文在东北地区多年平均的年均温、年降水分布图,海拔高程图、坡度图、坡向图和植被图的基础上,使用地理信息系统和Logistic回归模型的结合,预测3种落叶松(Larix sp.)的“气候-地形”潜在分布区。预测精度用敏感性、指定度和总正确率进行评价,3个树种的敏感性为61%~88%,指定度为80%~99.8%,总正确率为80%~99.8%。年均温、年降水和海拔是控制3种落叶松分布的主要环境因子。采用5种气温变化方案(+1 ℃、+2 ℃、+3 ℃、+4 ℃和+5 ℃)和6种降水变化方案(-30%、-20%、-10%、+10%、+20%和+30%),预测气候变化对各个树种潜在分布的影响,探索不同的树种对气候因子的敏感性。结果表明,气温每上升1 ℃,兴安落叶松(Larix gmelinii)将减少12%;长白落叶松(Larix olgensis var. changpaiensis)将增加23%;华北落叶松(Larix principis-rupprecntii)将增加500%。降水每增加10%,兴安落叶松将减少12.5%;长白落叶松将增加64%;华北落叶松将减少15%;随气候的“暖干化"(+5 ℃,-30%),兴安落叶松将向西北方退缩100 km左右;长白落叶松向西北方扩展100 km左右;华北落叶松将向东北方扩展800 km左右。随气候的“暖湿化"(+5 ℃,+30%),兴安落叶松将向西北退缩400 km左右;长白落叶松将向西北方扩展550 km;华北落叶松将向东北方扩展320 km左右。     

Atrophy and Other Potential Factors Affecting Long Term Deep Brain Stimulation Response: A Case Series

Objective

To describe three DBS cases which presented with new side effects or loss of benefit from stimulation after long-term follow-up and to discuss the potential contributing factors.

Methods

A University of Florida (UF) database (INFORM) search was performed, identifying three patients, two Parkinson''s disease (PD) and one Essential Tremor (ET), with an unexpected change in long-term programming thresholds as compared to initial evaluation. Clinical follow-up, programming, imaging studies, and lead measurements were reviewed. The UF Institutional Review Board (IRB) approved this study.

Results

A substantial increase in the 3^rd ventricular width (120%), Evans index (6%), ventricular index (5%), and cella media index (17%) was uncovered. A change in thresholds across lead contacts with a decrease in current densities as well as a relative lateral change of lead location was also observed. Hardware-related complications, lead migration, and impedance variability were not identified.

Conclusions

Potential factors contributing to long-term side effects should be examined during a DBS troubleshooting assessment. Clinicians should be aware that in DBS therapy there is delivery of electricity to a changing brain, and atrophy may possibly affect DBS programming settings as part of long-term follow-up.     

Psychological responses to genetic testing for weight gain: a vignette study

Genetic testing for obesity risk is increasingly available to the public but few studies have examined motivational or affective reactions. Here we report findings from a "vignette" study investigating reactions to "higher-risk" and "average-risk" results for the obesity-related FTO gene in two groups: a panel sample of individuals with weight concerns, for whom testing may have treatment implications (n = 306, mean age = 45 years, mean BMI = 35) and a student sample (n = 395, mean age = 25 years, mean BMI = 23), for whom testing would have implications for obesity prevention. Participants were given FTO gene information that described higher-risk alleles as linked with modest weight gain and slightly higher risk of obesity. They responded to both higher- and average-risk vignettes, with order randomized. Interest in genetic testing was high overall, and higher in panel respondents than students (93% vs. 78% would "probably" or "definitely" have the test; P < 0.001). In students, a higher-risk result generated higher motivation to change (d = 0.15; P < 0.001), but also slightly higher negative affect (d = 0.03, P < 0.001) and fatalism (d = 0.05, P < 0.001) than an average-risk result. Panel respondents also had higher motivation to change (d = 0.17, P < 0.001) as well as relief about having an explanation for their body weight (d = 0.02, P = 0.013) in the higher-risk condition, but no increase in fatalism or depression. These results suggest that at the level of anticipated responses to FTO gene feedback, higher-risk results had positive motivational effects with minimal changes in negative affect or fatalism. Genetic testing has the potential to be a useful clinical or preventive tool when combined with appropriate information.     

The Forgotten Component of Plant Water Potential: A Reply - Tissue Pressures are not Additive in the Way M. J. Canny Suggests

Abstract: Martin Canny's concepts of "tissue pressure" and its derivative "compensating pressure" are reviewed. Tissue pressure arises when the volume change of some living cells exerts a pressure on adjacent living or dead cells. Contrary to previous assertions, tissue pressure cannot cause a permanent change in pressure potential or water potential of adjacent cells. Tissue pressure induces only a transitory increase of pressure and water potential. After equilibrium is reestablished, the same or a more negative pressure or water potential results. The idea that tissue pressure can prevent or repair xylem embolism is without merit.     

Relationship Between Escherichia coli B Titer and the Level of Deoxycytidylate Deaminase Activity Induced on Bacteriophage T2r+ Infection

The activities of six bacteriophage T2r(+)-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coli B from 8 x 10(8) to 2 x 10(8) cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 x 10(8) to 8 x 10(8) per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen.     

Potential-dependent changes in ionic selectivity of batrachotoxin-modified sodium channels of frog nerve fiber

Ionic currents through sodium channels modified by batrachotoxin were measured by the voltage clamp method on a myelinated frog nerve fiber membrane. The reversal potential (E_rev) of steady-state currents was shown to be on the average 5 mV less positive than E_rev corresponding to the initial (peak) values of the currents. The results of control experiments using procaine and tetrodotoxin showed that the change in E_rev observed during a depolarizing pulse is not connected with the presence of unmodified sodium channels or unblocked potassium channels, with nonlinearity of leakage, or with a change in transmembrane gradients of current-carrving cations. In experiments with measurement of "instant" currents it was shown that E_rev becomes less positive as the amplitude and duration of preliminary depolarization increase. The results support the view that sodium-potassium selectivity of batrachotoxin-modified sodium channels depends on potential.     

Inhibition of TNFalpha in vivo prevents hyperoxia-mediated activation of caspase 3 in type II cells

Background

The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells).

Methods

Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits.

Results

In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells.

Conclusion

In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells.     

Role of interferon γ and tumour necrosis factor α in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line

Summary In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon (IFN) or tumour necrosis factor (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [³H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.