Accumulation of protein aggregates induces autolytic programmed cell death in hybrid tobacco cells expressing hybrid lethality.

Hybrid cells of Nicotiana suaveolens x N. tabacum grow normally at 36 °C, but immediately express lethality due to probable autoimmune response when transferred from 36 to 28 °C. Our recent study showed that the temperature-sensitive lethality of these hybrid cells occurs through autolytic programmed cell death (PCD). However, what happens in hybrid cells following the induction of autoimmune response to autolytic PCD is unclear. We hypothesized that accumulation of protein aggregates in hybrid cells induces autolytic PCD and examined detergent-insoluble protein (protein aggregates) isolated from hybrid cells expressing lethality. The amount of insoluble proteins increased in hybrid cells. Sodium 4-phenylbutyrate, a chemical chaperone, inhibited both the accumulation of insoluble proteins and irreversible progression of cell death. In contrast, E-64, a cysteine protease inhibitor, accelerated both the accumulation of insoluble proteins and cell death. Moreover, proteome analysis revealed that proteasome-component proteins were accumulated specifically in cells treated with E-64, and proteasome activity of hybrid cells decreased after induction of lethality. These findings demonstrate that accumulation of protein aggregates, including proteasome subunits, eventually cause autolytic PCD in hybrid cells. This suggests a novel process inducing plant PCD by loss of protein homeostasis and provides clues to future approaches for elucidating the whole process.

Acute change of titin at mid-sarcomere remains despite 8 wk of plyometric training.

The purpose of this study was to investigate skeletal muscle changes induced by an acute bout of plyometric exercise (PlyEx) both before and after PlyEx training, to understand if titin is affected differently after PlyEx training. Healthy untrained individuals (N = 11) completed the 1stPlyEx (10 × 10 squat-jumps, 1-min rest). Thereafter, six subjects completed 8 wk of PlyEx, while five controls abstained from any jumping activity. Seven days after the last training session, all subjects completed the 2ndPlyEx. Blood samples were collected before and 6 h and 1, 2, 3, and 4 days after each acute bout of PlyEx, and muscle biopsies 4 days before and 3 days after each acute bout of PlyEx. The 1stPlyEx induced an increase in circulating myoglobin concentration. Muscle sample analysis revealed Z-disk streaming, a stretch or a fragmentation of titin (immunogold), and increased calpain-3 autolysis. After training, 2ndPlyEx did not induce Z-disk streaming or calpain-3 activation. The previously observed post-1stPlyEx positional change of the titin COOH terminus was still present pre-2ndPlyEx, in all trained and all control subjects. Only two controls presented with Z-disk streaming after 2ndPlyEx, while calpain-3 activation was absent in all controls. Eccentric explosive exercise induced a stretch or fragmentation of titin, which presented as a positional change of the COOH terminus. Calpain-3 activation does not occur when titin is already stretched before explosive jumping. Enzymatic digestion results in titin fragmentation, but since an increase in calpain-3 autolysis was visible only after the 1stPlyEx acute bout, fragmentation cannot explain the prolonged positional change.

Neurine, an acetylcholine autolysis product, elevates secreted amyloid-beta protein precursor and amyloid-beta peptide levels, and lowers neuronal cell viability in culture: a role in Alzheimer's disease?

Classical hallmarks of Alzheimer's disease (AD) are a synaptic loss, cholinergic neuron death, and abnormal protein deposition, particularly of toxic amyloid-beta peptide (Abeta) that is derived from amyloid-beta protein precursor (AbetaPP) by the action of beta- and gamma-secretases. The trigger(s) initiating the biochemical cascades that underpin these hallmarks have yet to be fully elucidated. The typical forebrain cholinergic cell demise associated with AD brain results in a loss of presynaptic cholinergic markers and acetylcholine (ACh). Neurine (vinyl-trimethyl-ammonium hydroxide) is a breakdown product of ACh, consequent to autolysis and is an organic poison found in cadavre brain. The time- and concentration-dependent actions of neurine were assessed in human neuroblastoma (NB, SK-N-SH) cells in culture by quantifying cell viability by lactate dehydrogenase (LDH) and MTS assay, and AbetaPP and Abeta levels by Western blot and ELISA. NB cells displayed evidence of toxicity to neurine at > or = 3 mg/ml, as demonstrated by elevated LDH levels in the culture media and a reduced cell viability shown by the MTS assay. Using subtoxic concentrations of neurine, elevations in AbetaPP and Abeta1-40 peptide levels were detected in conditioned media samples.

Enzymatic versus autolytic debridement of chronic leg ulcers: a prospective randomised trial.

OBJECTIVE: A randomised clinical trial (n = 42) compared the effectiveness of two approaches to debriding chronic leg ulcers: TenderWet 24, which is designed to support the autolytic degradation process, and Iruxol N (Santyl), an enzymatic treatment claimed to enhance the degradation process. METHOD: Patients were randomly assigned to one of the two treatment groups for three weeks. Wounds were evaluated weekly for the amount of eschar/slough, the area of healthy granulation and the re-epithelialised area. RESULTS: During days 1-14 slough within the groups was reduced by almost 19% for TenderWet 24 and by 9% for Iruxol N, followed by an increase of 26% and 10% respectively in granulation tissue. These effects were less accentuated during days 7-21. There was a further 11% improvement in tissue debridement for the TenderWet 24 group and a relapse (+9.1%) in the Iruxol N group. CONCLUSION: Although TenderWet 24 appeared to be more efficient in a few cases, the general efficacy of the two products appeared to be almost the same as no statistically significant superiority of either product was found.

Temperature-correction of abdominal impedance: improved relationship between impedance and postmortem interval.

The relationship between extracellular abdominal impedance and postmortem interval (PMI) reflects the combined effects, on impedance, of postmortem cooling of the tissues and of autolysis per se. This study was performed in order to eliminate temperature change as a major factor contributing to the time course of postmortem change in abdominal impedance. Dissociation of thermal and autolytic influences was achieved by recording deep abdominal temperature at the time of impedance measurement, followed by correction of all measured impedances to their theoretically predicted values at an arbitrarily chosen temperature of 40 degrees C. Uncorrected abdominal impedance increased from 82+/-12 Ohmz, 1 h after death, to 108+/-21 Ohmz after 12 h. Impedance then decreased to 96+/-23, 89+/-22, 75+/-19, 66+/-21 and 59+/-19 Ohmz at postmortem intervals of 24, 36, 48, 60 and 72 h, respectively. In contrast, corrected abdominal impedance decreased progressively from 63+/-7 Ohmz, 1 h after death, to 61+/-9, 56+/-11, 51+/-10, 46+/-10, 39+/-11 and 35+/-10 Ohmz at postmortem intervals of 12, 24, 36, 48, 60 and 72 h, respectively. The improved relationship between (corrected) abdominal impedance and PMI is of potential value in estimating time since death.

Mechanism and role of trypsinogen activation in acute pancreatitis.

In healthy subjects, the 3 known pancreatic trypsinogens, which are endopeptidases belonging to the chymotrypsin superfamily, are activated by enterokinase and partial autoactivation in the duodenum. The premature activation of trypsinogen in the pancreatic interstitium, with the subsequent activation of other pancreatic zymogens, is believed to lead to the autodigestion of the gland, this being the first event in acute pancreatitis. The mechanisms that lead to trypsinogen, activation in acute pancreatitis are largely unknown. However, ischemia, hypercalcemia and the activation of cathepsin B (by cholecystokinin) are thought to be of importance. The easiest and most reliable way to assess trypsinogen activation is the measurement of the activation peptide, TAP, in urine, plasma, pancreatic tissue or ascitic fluid. In the animal model of acute pancreatitis, TAP in ascites and pancreatic tissue has been shown to correlate with the presence and extent of necroses. It has proven to be a good marker for the severity of pancreatitis and is a useful marker in examining the pathophysiology and possible treatment modalities in the animal model of acute pancreatitis. Studies on TAP in human acute pancreatitis were most commonly focused on urinary TAP. Within a 48-hour time frame after the onset of the disease, TAP was a good predictor of the severity of acute pancreatitis. The main advantage over other markers, such as CRP, is that TAP is the earliest marker of necrosis to be increased. Also, increased levels of TAP in ascitic fluid were shown to correlate well with pancreatic necroses. In our experience, plasma TAP was found to have a "diagnostic window" within the first 3 days predicting pancreatic necroses. Positive TAP gave a very good positive prediction and a high specificity towards the development of pancreatic necroses, but did not differ between necrotizing pancreatitis with systemic complications or uncomplicated necrotizing pancreatitis. We therefore think that plasma TAP is a very good marker for local complication in acute pancreatitis and its routine measurements may help to identify patients at a high risk within the first days of the disease.

The role of calcium in pancreatitis.

BACKGROUND/AIMS: A large, sustained increase in acinar [Ca2+]i may play a key role in the pathogenesis of acute pancreatitis. Many mechanisms which lead to cell damage in vitro and pancreatitis in vivo, such as free radicals or supraphysiological cerulein concentrations, cause a rapid increase in [Ca2+]i in pancreatic acinar cells. Little is known about why [Ca2+]i increases in some instances stimulate secretion and in other instances initiate cell death. So far, [Ca2+]i increases were thought to represent physiological signals when they occurred as oscillations at the single cell level. METHODOLOGY: This paper reviews recent literature and our own original research about the role of calcium in the function of pancreatic acinar cells and the development of pancreatitis. RESULTS: Recent studies showed that exposure of acinar cells to free radicals not only caused a bulk increase in [Ca2+]i but also resulted in calcium oscillations which had a lower frequency than, but similar amplitude to oscillations occurring after physiological stimuli. The absolute increase in [Ca2+]i did not definitely determine the cellular response. Instead, the duration of [Ca2+]i increase may have been more important. In contrast to previous belief of a direct relationship between [Ca2+]i oscillations and exocytosis, recent results show that radicals can induce [Ca2+]i oscillations which do not exert exocytosis but inhibit the secretory response to physiological stimuli. Further experiments showed that the [Ca2+]i release caused by radicals originates from thapsigargin-insensitive, ryanodine-sensitive stores. CONCLUSIONS: The origin and duration of [Ca2+]i increases rather than their extent or oscillatory nature, determine whether the cell will secrete or die. An abnormal [Ca2+]i increase can trigger trypsin activation, acinar cell damage and acute pancreatitis. This hypothesis is supported by studies which show that calcium chelators inhibit radical-induced trypsin activation as well as cell necrosis and apoptosis. Thus, an inhibition of pathological [Ca2+]i release may have a therapeutic potential.

Changes in cadaveric cancellous vertebral bone strength in relation to time. A biomechanical investigation.

STUDY DESIGN: A biomechanical study was conducted during a 3.5-day period to test for changes occurring in pullout strengths of cancellous screws inserted into human cadaveric vertebral bodies. OBJECTIVES: To quantify, within the testing time of 3.5 days, the possible changes to the mechanical properties of cadaveric vertebral bodies, resulting from structural degradation caused by postmortem, time-dependent, autolytic processes during mechanical testing of implant-bone biomechanics. SUMMARY OF BACKGROUND DATA: Biomechanical testing of whole spinal implants and analysis of the screw-bone interface of spinal implants is an area of clinical interest that frequently requires the use of cadaveric spine specimens. Changes in vertebral bone properties during the testing period may invalidate experimental results, but no data are available on degradation of bone during the testing period. METHODS: Anterior oblique cancellous screws were inserted into human vertebral bodies from which the ventral cortex had been removed. The pullout strength was measured at 0, 24, 60, and 84 hours after insertion. The tests were performed on 48 human vertebral bodies, which were stored by freezing to -23 C, thawed for testing, and kept at room temperature during the testing time for as long as 84 hours. RESULTS: The axial pullout strength showed no statistically significant change during 84 hours (P = 0.15). There were no significant differences attributable to vertebral level from T4 to L4, probably because the ventral cortices had been removed (P = 0.7). CONCLUSIONS: During 3.5 days, there were no changes in pullout strength of vertebral cancellous bone. In biomechanical studies during a maximum period of 3 days with a small number of cadaveric spines (e.g., four spine specimen) the time-dependent changes in pullout strength play a less significant role than do the interspine differences. Interspine differences should be regarded as an important factor to be considered in the design of biomechanical tests.

Adipocere--problems in estimating the length of time since death.

Establishing the length of time since death is particularly difficult in corpses showing advanced autolysis. One sign of advanced decay is the formation of adipocere. In this state bodies show evidence of a partly wax-like and partly pasty condition. Continued storage ultimately results, among other things, in further decomposition due to the action of micro-organisms from the surrounding area-even if this is chronologically delayed. An exception is provided by the formation of adipocere under air-tight conditions. Initially, autolysis and heterolysis also occur, involving the release of fatty acids. As a result of the subsequent hydrogenation of the fats under the influence of bacterial enzymes, the unsaturated fatty acids are partially converted into saturated fatty acids. As the fatty acids clearly have a bactericidal effect, further bacterial decomposition is stopped at this early adipocere stage. Additional micro-organisms from outside can no longer penetrate when this hermetic seal is in place. In addition, the lack of calcium (e.g. from water or moist earth) can be a reason for the fact that further adipocere development, leading to wax-like hardening of the fat, is arrested. Thus the condition of the body can remain constantly preserved over many years and it no longer allows a reliable estimate to be made of the period of time since death.

Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum.

Salivary gland degeneration in ixodid ticks is triggered by an ecdysteroid hormone. We used [3H]ponasterone A (PoA) as a specific ligand to detect the ecdysteroid receptor in the salivary glands of large, partially fed female ticks (Amblyomma hebraeum Koch; Acari: Ixodidae). Binding of [3H]PoA was thermolabile and sensitive to pronase, but not to DNase or RNase, indicating that the ligand binds to a protein. Scatchard analysis of [3H]PoA binding strongly suggested the presence of an ecdysteroid receptor in cytosolic and nuclear extracts of the tissue. The Kd and Bmax for PoA binding in cytosol were 0.72 +/- 0.09 nM and 175 +/- 12 fmol/mg protein, respectively (n = 8). Corresponding figures for nuclear extract were 1.1 +/- 0.5 nM and 282 +/- 35 fmol/mg protein, respectively (n = 3; P > 0.05 compared to cytosol). The relative ability of unlabeled ecdysteroids to compete for [3H]PoA binding was (in descending order): PoA > muristerone A > makisterone A > 20-hydroxyecdysone > mesylinokosterone > ecdysone. The Kd estimated for 20-hydroxyecdysone (probably the natural hormone) correlates very well with its physiological potency in inducing salivary gland degeneration in vivo and in organ culture. None of the vertebrate steroids tested (estradiol, testosterone, progesterone, and corticosterone) was able to displace PoA binding at a concentration 10(5) times higher than PoA. The cytosolic form of the receptor migrated to the 3.2 S region of a 10-40% sucrose density gradient.

[Cardioprotective effects of adaptation to restraint stress and hypoxia]. / Kardioprotektornye éffekty adaptatsii k immobilizatsionnym stressam i gipoksii.

Isolated rat heart experiments have revealed that restraint stress adaptation results in enhanced resistance of the isolated heart to reperfusion. There is also a higher resistance to the autolysis of the organelles isolated from the hearts of stress-adapted animals. This complex of changes is designated as a phenomenon of adaptive stabilization of structures (PhASS). The phenomenon developing in restraint stress adaptation substantially limits arrhythmias, contracture, contraction amplitude depression, and creatine kinase release into the perfusate in thermal damage to the isolated rat heart. Simultaneously, PhASS is accompanied by a multiple increase in five hsp70 isoforms with pI 5.8-6.3 in cytosol and two isoforms with pI about 6.3 in the nucleoplasm. Only two hsp70 isoforms with pI about 5.8 accumulate solely in cytosol during adaptation to intermittent hypoxia. Consistently, the resistance of Ca(2+)-pump and nuclear DNA remains unchanged and the protection against reperfusion and thermal damage are several times less pronounced.

Factors affecting the loss of mitochondrial function in autolyzing cardiac muscle.

Rates of loss of mitochondrial respiratory function were monitored during autolyses of canine myocardial samples pretreated so as to affect tissue pH and/or tissue ATP content prevailing during tissue autolysis. When autolyses occurred under conditions of differing tissue pH, but at nearly identical tissue ATP levels, the rate of loss of mitochondrial function was virtually unchanged suggesting that tissue acidosis in the absence of a concomitant tissue ATP differential had little or no effect upon the rate of progression of mitochondrial damage. In a second comparison, autolyses were carried out at constant tissue pH, but where tissue ATP content differed dramatically. Here, the rate of loss of mitochondrial function was increased markedly suggesting that tissue ATP depletion in the absence of a concomitant tissue pH differential had a major effect upon the rate of loss of mitochondrial function. Thus, of the two parameters studied, tissue ATP content alone was far more important than tissue pH alone in determining the rate of cell membrane damage during ischemia. Finally, autolyses were carried out where both tissue pH and ATP content differed. Here, an even more dramatic increase in the rate of progression of mitochondrial damage occurred suggesting the operation of synergism between tissue ATP depletion and acidosis in promoting cell injury in ischemic cardiac muscle.

ONO-3144, a new anti-inflammatory drug and its possible mechanism of action.

Anti-inflammatory activities of ONO-3144 have been studied in various experimental models. This compound showed an inhibitory effect on increased vascular permeability and acute inflammation. In the carrageenin test the activity of ONO-3144 was comparable to that of indomethacin (IM), while in the dextran, albumin, yeast and scald edema test it was more potent than that of IM. Unlike IM, phenylbutazone (PB) and tiaramide (TI), ONO-3144 showed marked inhibitory effects on H2O2-induced hemolysis and lipid peroxidation. In the prostaglandin (PG) biosynthesis series, ONO-3144 did not inhibit cyclooxygenase activity but stimulated PG hydroperoxidase activity, facilitated conversion to PGH2 and also inhibited thromboxane (Tx) synthetase. The findings suggest that ONO-3144 is potentially a new type of anti-inflammatory drug. Possible sites of action of ONO-3144 are discussed.

3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes.

3-Methyladenine (5 mM) inhibits endogenous protein degradation in isolated rat hepatocytes by about 60%, while having no adverse effect on the degradation of an exogenous protein (asialofetuin), on protein synthesis, or on intracellular ATP levels. 3-Methyladenine appears to act specifically upon the autophagic/lysosomal pathway of degradation, as judged from its lack of effect in the presence of amino acids or a lysosomotropic amine (propylamine). The effect of the purine is not mediated by amino acids because the inhibition of protein degradation is accompanied by a significant depression of intracellular amino acid levels. The ability of 3-methyladenine to suppress the formation of electron microscopically visible autophagosomes suggests that it may be regarded as a specific inhibitor of autophagy.

Changes in energy transduction efficiency in mitochondria isolated from ischemic myocardium.

The effects of temporary coronary artery occlusion (TCAO) in vivo and ischemia in situ (autolysis) on mitochondrial functions and inner membrane permeability were investigated. It was found that one day after TCAO the permeability of inner membrane to H+ was increased 64% and the maximal activity of the glutamate plus malate oxidase system was reduced 35% with respect to control. The proton-electrochemical gradient and the rate of oxidative phosphorylation were decreased 35% and 55%, respectively. The most significant alterations during autolysis were observed in respiratory control ratio, state IV respiration and in H+ permeability of inner membrane. The H+ permeability of inner membrane increased progressively with time, and after 4 hr of autolysis it was 378% higher than the control. The data suggest that the alterations in energy transduction efficiency of mitochondria isolated from ischemic myocardium depend largely on the increase of inner membrane permeability to H+.

Demonstration of the physiological role of autolysis by a comparative study with a wild-type and its non-autolytic mutant of Micrococcus lysodeikticus (luteus) cultivated with externally added proteolytic enzymes.

The log phase cells of autolytic Microccus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced. A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology: large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells. The possibility that there are two kinds or phases of autolytic enzymes "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.

[Investigations of the autolysis of liver by passive electrical and thermodynamic statistical methods (author's transl)]. / Passiv-elektrische und thermodynamisch-statistische Untersuchungen der Autolyse von Lebergewebe

From continuous passive-electrical measurements on autolyzing liver tissue are derived statements about the structural changes in time. In the early post mortem phase the autolysis is a cell-controled process, which suddenly goes over in an uncontroled degradation process. The changes of the uncontroled process are fitted. The results are statistically and thermodynamically interpreted by a condensation modell. The high complexity of the living resp. cell-controled system correlates to a relatively high structural entropy.