Impaired brain creatine kinase activity in Huntington's disease.

BACKGROUND: Huntington's disease (HD) is associated with impaired energy metabolism in the brain. Creatine kinase (CK) catalyzes ATP-dependent phosphorylation of creatine (Cr) into phosphocreatine (PCr), thereby serving as readily available high-capacity spatial and temporal ATP buffering. OBJECTIVE: Substantial evidence supports a specific role of the Cr/PCr system in neurodegenerative diseases. In the brain, the Cr/PCr ATP-buffering system is established by a concerted operation of the brain-specific cytosolic enzyme BB-CK and ubiquitous mitochondrial uMt-CK. It is not yet established whether the activity of these CK isoenzymes is impaired in HD. METHODS: We measured PCr, Cr, ATP and ADP in brain extracts of 3 mouse models of HD - R6/2 mice, N171-82Q and HdhQ(111) mice - and the activity of CK in cytosolic and mitochondrial brain fractions from the same mice. RESULTS: The PCr was significantly increased in mouse HD brain extracts as compared to nontransgenic littermates. We also found an approximately 27% decrease in CK activity in both cytosolic and mitochondrial fractions of R6/2 and N171-82Q mice, and an approximately 25% decrease in the mitochondria from HdhQ(111) mice. Moreover, uMt-CK and BB-CK activities were approximately 63% lower in HD human brain samples as compared to nondiseased controls. CONCLUSION: Our findings lend strong support to the role of impaired energy metabolism in HD, and point out the potential importance of impairment of the CK-catalyzed ATP-buffering system in the etiology of HD.

Evolution of a multidisciplinary clinical pathway for the management of unstable patients with pelvic fractures.

OBJECTIVE: To determine whether the evolution of the authors' clinical pathway for the treatment of hemodynamically compromised patients with pelvic fractures was associated with improved patient outcome. SUMMARY BACKGROUND DATA: Hemodynamically compromised patients with pelvic fractures present a complex challenge. The multidisciplinary trauma team must control hemorrhage, restore hemodynamics, and rapidly identify and treat associated life-threatening injuries. The authors developed a clinical pathway consisting of five primary elements: immediate trauma attending surgeon's presence in the emergency department, early simultaneous transfusion of blood and coagulation factors, prompt diagnosis and management of associated life-threatening injuries, stabilization of the pelvic girdle, and timely insinuation of pelvic angiography and embolization. The addition of two orthopedic pelvic fracture specialists led to a revision of the pathway, emphasizing immediate emergency department presence of the orthopedic trauma attending to provide joint decision making with the trauma surgeon, closing the pelvic volume in the emergency department, and using alternatives to traditional external fixation devices. METHODS: Using trauma registry and blood bank records, the authors identified pelvic fracture patients receiving blood transfusions in the emergency department. They analyzed patients treated before versus after the May 1998 revision of the clinical pathway. RESULTS: A higher proportion of patients in the late period had blood pressure less than 90 mmHg (52% vs. 35%). In the late period, diagnostic peritoneal lavage was phased out in favor of torso ultrasound as a primary triage tool, and pelvic binding and C-clamp application largely replaced traditional external fixation devices. The overall death rate decreased from 31% in the early period to 15% in the later period, as did the rate of deaths from exsanguination (9% to 1%), multiple organ failure (12% to 1%), and death within 24 hours (16% to 5%). CONCLUSIONS: The evolution of a multidisciplinary clinical pathway, coordinating the resources of a level 1 trauma center and directed by joint decision making between trauma surgeons and orthopedic traumatologists, has resulted in improved patient survival. The primary benefits appear to be in reducing early deaths from exsanguination and late deaths from multiple organ failure.

Chemosensory responses of Tetrahymena thermophila to CB2, a 24-amino-acid fragment of lysozyme.

While lysozyme is a depolarizing chemorepellent in Tetrahymena, the entire lysozyme molecule is not necessary to activate the lysozyme receptor. Reduced lysozyme was cut into three fragments by cyanogen bromide cleavage and the fragments (CB1, CB2 and CB3) were separated by HPLC. Behavioral bioassays showed that the carboxy-terminal 24-amino-acid fragment, which we call CB2, is 100 times more active than intact lysozyme as a chemorepellent. CB2 appears to activate the same receptor as lysozyme because behavioral cross-adaptation is seen between these two compounds and an antibody generated to the purified lysozyme receptor blocks responses to both lysozyme and CB2. This is further supported by the observation that neomycin, which is a competitive inhibitor of lysozyme binding, also inhibits CB2 responses. This inhibition may be due to the fact that neomycin is highly positively charged (+5 at pH 7.0) and CB2 has a net charge of +4 at pH 7.0. Intracellular electrophysiological recordings documented that CB2 elicits a transient, depolarizing receptor potential that is similar to the lysozyme-induced depolarizations except they are much smaller. CB2 is a more potent and specific ligand for use in studies of the lysozyme receptor of Tetrahymena.

Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis.

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death-inducing stimulus. We demonstrate that the activation of caspase-3, in Jurkat cells stimulated to undergo apoptosis by a Fas-independent pathway, is catalyzed by caspase-6. Caspase-6 was found to co-purify with caspase-3 as part of a multiprotein activation complex from extracts of camptothecin-treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase-3 could be demonstrated by co-immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase-3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP-dependent 'foldase' activity of Hsp60 improves the vulnerability of pro-caspase-3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.

Purification and characterization of a novel chemorepellent receptor from Tetrahymena thermophila.

Chemosensory transduction and adaptation are important aspects of signal transduction mechanisms in many cell types, ranging from prokaryotes to differentiated tissues such as neurons. The eukaryotic ciliated protozoan, Tetrahymena thermophila, is capable of responding to both chemoattractants (O'Neill et al., 1985; Leick, 1992; Kohidai, Karsa & Csaba, 1994, 1995) and chemorepellents (Francis & Hennessey, 1995; Kuruvilla, Kim & Hennessey, 1997). An example of a nontoxic, depolarizing chemorepellent in Tetrahymena is extracellular lysozyme (Francis & Hennessey, 1995; Hennessey, Kim & Satir, 1995). Lysozyme is an effective chemorepellent at micromolar concentrations, binds to a single class of externally facing membrane receptors and prolonged exposure (10 min) produces specific chemosensory adaptation (Kuruvilla et al., 1997). We now show that this lysozyme response is initiated by a depolarizing chemoreceptor potential in Tetrahymena and we have purified the membrane lysozyme receptor by affinity chromatography of solubilized Tetrahymena membrane proteins. The solubilized, purified protein is 42 kD and it exhibits saturable, high affinity lysozyme binding. Polyclonal antibodies raised against this 42 kD receptor block the in vivo lysozyme chemoresponse. This is not only the first time that a chemoreceptor potential has been recorded from Tetrahymena but also the first time that a chemorepellent receptor has been purified from any unicellular eukaryote.

Extracellular GTP causes membrane-potential oscillations through the parallel activation of Mg2+ and Na+ currents in Paramecium tetraurelia.

Paramecium tetraurelia responds to extracellular GTP (>/= 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either Mg2+ or Na+ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar to that of the membrane-potential and behavioral responses. These currents were also Mg2+- and Na+-dependent, suggesting that GTP acts through Mg2+-specific (IMg) and Na+-specific (INa) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg2+ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks IMg ("eccentric"), while Na+ failed to support GTP responses in "fast-2," a mutant that specifically lacks INa. Both mutants responded normally to GTP if the alternative cation was provided. As IMg and INa are both Ca2+-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca2+ concentration. Indeed, applying GTP to cells in the absence of either Mg2+ or Na+ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known voltage-dependent Ca2+ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor-coupled Ca2+ conductance.

A soluble ecto-ATPase from Tetrahymena thermophila: purification and similarity to the membrane-bound ecto-ATPase of smooth muscle.

For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote, Tetrahymena, at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme in Tetrahymena. It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+ or Mg2+ dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain, N-ethylmaleimide and p-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ecto-ATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein in Tetrahymena plasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase of Tetrahymena may play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.

beta-Sitosterol inhibits HT-29 human colon cancer cell growth and alters membrane lipids.

The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary phytosterol on the growth of HT-29 cells, a human colon cancer cell line. In addition, the incorporation of this phytosterol into cellular membranes and how this might influence the lipid composition of the membranes were investigated. Tumor cells were grown in DMEM containing 10% FBS and supplemented with sterols (cholesterol or beta-sitosterol) at final concentrations up to 16 microM. The sterols were supplied to the media in the form of sterol cyclodextrin complexes. The cyclodextrin used was 2-hydroxypropyl-beta-cyclodextrin. The sterol to cyclodextrin molar ratio was maintained at 1:300. The study indicated that 8 and 16 microM beta-sitosterol were effective at cel growth inhibition as compared to cholesterol or to the control (no sterol supplementation). After supplementation with 16 microM beta-sitosterol for 9 days, cell growth was only one-third that of cells supplemented with equimolar concentration of cholesterol. No effect was observed on total membrane phospholipid concentration. At 16 microM beta-sitosterol supplementation, membrane cholesterol was reduced by 26%. Cholesterol supplementation resulted in a significant increase in the cholesterol/phospholipid ratio compared to either beta-sitosterol supplemented cells or controls. There was a 50% reduction in membrane sphingomyelin (SM) of cells grown in 16 microM beta-sitosterol. Additional changes were observed in the fatty acid composition of minor phospholipids of beta-sitosterol supplemented cells, such as SM, phosphatidylserine (PS), and phosphatidylinositol (PI). Only in the case of PI, was there an effect of these fatty acid changes on the unsaturation index, beta-sitosterol incorporation resulted in an increase in the U.I. It is possible that the observed growth inhibition by beta-sitosterol may be mediated through the influence of signal transduction pathways that involve membrane phospholipids.

Lysozyme acts as a chemorepellent and secretagogue in Paramecium by activating a novel receptor-operated Ca++ conductance.

Using combined intracellular recordings and behavioral bioassays, it was found that lysozyme has two different effects in Paramecium, depending upon the concentrations used. At low concentrations (0.5 mM to 1.0 microM) it acts as an effective chemorepellent that causes reliable electrophysiological changes. Lysozyme-induced somatic depolarizations, isolated by blocking K+ channels with Cs-TEA, showed concentration dependencies that were well correlated with chemorepulsion. Ion dependency experiments showed that these were Ca++ based depolarizations. Addition of either Na+ or Mg++ improves chemorepulsion by providing additional depolarizations. Both the depolarizations and chemorepulsion were blocked by 10 microM neomycin, suggesting that the depolarization is necessary for this chemosensory transduction event. At higher concentrations (100 microM), lysozyme is a secretagogue. A transient inward current, recorded in Ca++ alone solutions with Cs-TEA present, was seen in response to high lysozyme concentrations. The amplitude of this inward current was well correlated with exocytosis. Addition of neomycin (1.0 mM) eliminated both the inward current and exocytosis, suggesting a causal relationship. Neither amiloride or W-7, compounds previously suggested to affect the electrophysiological responses to secretagogues, had any significant effects. The mucopolysaccharide hydrolysis activity of lysozyme was not required for any of these responses. We propose that Paramecium have a high affinity receptor on the body plasma membrane that responds to either lysozyme or a related compound to cause an increase in a novel body Ca++ conductance. This receptor-operated Ca++ conductance causes membrane depolarization and chemorepulsion at low concentrations and triggers a sufficient Ca++ influx at high concentrations to cause exocytosis.

Chemorepellents in Paramecium and Tetrahymena.

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.

Oxidants act as chemorepellents in Paramecium by stimulating an electrogenic plasma membrane reductase activity.

Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptation and cellular sensory integration. While millimolar amounts of many attractants hyperpolarize and cause faster forward swimming, oxidants are repellents that depolarize and cause backward swimming at micromolar concentrations. The non-permeant oxidants cytochrome c, nitro blue tetrazolium and ferricyanide are repellents with half maximal concentrations of 0.4 microM, 2.2 microM and 100 microM respectively. In vivo reductase activities follow the same order of potencies. The concentration dependence of the cytochrome c reductase activity is well correlated with cytochrome c-induced depolarizations. This suggests that plasma membrane reduction of external cytochrome c is electrogenic, causing membrane depolarization and chemorepulsion. The reductase activity also appears to be voltage dependent. Depolarization by either K+, Na+, Ca++ or Mg++ correlates with inhibition of both in vivo reductase activities and cytochrome c-induced membrane potential changes. These responses were also seen in deciliated cells, showing that the body plasma membrane is sufficient for the response. Both chloroquine and diphenyleneiodonium inhibited reductase activities but only at unusually high concentrations. This activity showed no pH dependence in the physiological range. We propose that a plasma membrane bound NA-DPH-dependent reductase controls oxidant-induced depolarizations and consequent chemorepulsion.

External GTP alters the motility and elicits an oscillating membrane depolarization in Paramecium tetraurelia.

Paramecium, a unicellular ciliated protist, alters its motility in response to various stimuli. Externally added GTP transiently induced alternating forward and backward swimming interspersed with whirling at a concentration as low as 0.1 microM. ATP was 1000-fold less active, whereas CTP and UTP produced essentially no response. The response to the nonhydrolyzable GTP analogs guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta, gamma-imido]triphosphate was indistinguishable from that to GTP. This behavioral response was correlated with an unusual transient and oscillating membrane depolarization in both wild-type cells and the mutant pawn B, which is defective in the voltage-dependent Ca2+ current required for action potentials. This is a specific effect of external GTP on the excitability of a eukaryotic cell and, to our knowledge, is the first purinergic effect to be discovered in a microorganism.

Body plasma membrane vesicles from Paramecium contain a vanadate-sensitive Ca(2+)-ATPase.

Paramecia are an excellent model system for studying the mechanisms involved in sensory transductions and intracellular Ca2+ regulation. These cells have two functionally distinct plasma membrane domains, body and cilia. The body plasma membrane is responsible for transduction of sensory stimuli into receptor potentials and the ciliary membrane is required for Ca2+ action potentials. Although ciliary membrane vesicles (cmv) have been purified and well characterized, body plasma membranes have not. We have generated body plasma membrane vesicles (bmv) by homogenization of deciliated cells and purified them from the microsome fraction by a two-phase aqueous polymer separation. The major criteria for purity of the bmv fraction are: (i) It is enriched 15-fold for a known plasma membrane marker (immobilization antigen) while the marker activities for other membranes were all decreased. The protein banding pattern of bmv is generally similar to cmv on SDS-PAGE. (ii) It contains a vanadate-sensitive Ca(2+)-ATPase activity that has been suggested to be a plasma membrane Ca2+ pump. The specific activity of this bmv Ca(2+)-ATPase is increased 4-fold over that of the homogenate. (iii) The phospholipid, fatty acid, and sterol composition of the bmv fraction are indicative of plasma membranes because they are qualitatively similar to cmv. The bmv also contains a membrane-bound NADPH-dependent cytochrome c reductase activity, suggesting that it may play a role in body plasma membrane function. This purified bmv preparation is useful for studying the role of the body plasma membrane in Ca2+ regulation, sensory transduction, protein and lipid trafficking, and plasma membrane fusion events.

Environmental effects on circadian rhythms in photosynthesis and stomatal opening.

Persistent circadian rhythms in photosynthesis and stomatal opening occurred in bean (Phaseolus vulgaris L.) plants transferred from a natural photoperiod to a variety of constant conditions. Photosynthesis, measured as carbon assimilation, and stomatal opening, as conductance to water vapor, oscillated with a freerunning period close to 24 h under constant moderate light, as well as under light-limiting and CO2-limiting conditions. The rhythms damped under constant conditions conducive to high photosynthetic rates, as did rates of carbon assimilation and stomatal conductance, and this damping correlated with the accumulation of carbohydrate. No rhythm in respiration occurred in plants transferred to constant darkness, and the rhythm in stomatal opening damped rapidly in constant darkness. Damping of rhythms also occurred in leaflets exposed to constant light and CO2-free air, demonstrating that active photosynthesis and not simply light was necessary for sustained expression of these rhythms.

Evidence of multiple circadian oscillators in bean plants.

Circadian rhythms in stomatal opening and photosynthesis had shorter free-running periods than circadian rhythms in leaflet movement in bean plants (Phaseolus vulgaris L.) transferred from 12-hr photoperiods to constant conditions. The rhythm in leaflet movement had a period close to 27 hr, whereas the rhythm in stomatal opening, measured as conductance to water vapor, had a period close to 24 hr. Photosynthesis, measured as net assimilation of CO2, also oscillated with a period close to 24 hr. The periods of these rhythms did not vary with increasing temperature, demonstrating temperature compensation of the controlling oscillators. The difference in free-running periods displayed by these rhythms is evidence that multiple oscillators with different intrinsic frequencies operate in bean plants.

Covalent binding of lipids to cytoskeletal proteins of mouse mammary epithelial cells.

Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. Primary cultures of MMEC were incubated in the presence of 3H-palmitate for 4 h. A cytoskeletal (CS) fraction was prepared by treatment of the cells with 1.5M KCl and 1% Triton X-100. The residual material, consisting primarily of keratin and actin filaments was exhaustively (10-20 rounds, including sonications) extracted with chloroform/methanol to remove non-covalently bound labeled lipids. The CS protein was then acid-hydrolyzed and the chloroform-soluble products subjected to thin layer chromatography (TLC). Two-thirds of the covalently bound radiolabel appeared as a very hydrophobic peak on a TLC system optimized for separation of neutral lipids. Ten percent separated into 4-5 peaks on a polar lipid TLC system. A small amount of label was traced to fatty acid-like components. Autoradiography of two-dimensional gels indicated that all the CS proteins resolvable by Coomassie blue staining were also radiolabeled. The results are discussed in terms of CS-lipid-membrane interactions.

An intragenic suppressor of a calmodulin mutation in Paramecium: genetic and biochemical characterization.

We describe a suppressor of the calmodulin mutant cam1 in Paramecium tetraurelia. The cam1 mutant, which has a SER----PHE change at residue 101 of the third calcium-binding domain, inhibits the activity of the Ca(2+)-dependent K+ current and causes exaggerated behavioral responses to most stimuli. An enrichment scheme, based on an increased sensitivity to Ba2+ in cam1 cells, was used to isolate suppressors. One such suppressor, designated cam101, restores both the activity of the Ca(2+)-dependent K+ current and behavioral responses of the cells. We show that the cam101 mutant is an intragenic suppressor of cam1, based on genetic and microinjection data. The cam101 calmodulin is shown to be similar to wild-type calmodulin in terms of its ability to stimulate calmodulin-dependent phosphodiesterase at low concentrations of free calcium. However, the cam101 calmodulin has a reduced affinity for a monoclonal antibody to wild-type Paramecium calmodulin, as does the parental cam1 calmodulin, and a different mobility on acid-urea gels relative to both wild-type and cam1 calmodulin. We have been able to demonstrate that the isolation of intragenic suppressors of a calmodulin mutation is possible, which allows for the further genetic analysis of structure-function relationships in the calmodulin molecule.

Biochemical Correlates of the Circadian Rhythm in Photosynthesis in Phaseolus vulgaris.

A circadian rhythm in photosynthesis occurs in Phaseolus vulgaris after transfer from a natural or artificial light:dark cycle to constant light. The rhythm in photosynthesis persists even when intercellular CO(2) partial pressure is held constant, demonstrating that the rhythm in photosynthesis is not entirely due to stomatal control over the diffusion of CO(2). Experiments were conducted to attempt to elucidate biochemical correlates with the circadian rhythm in photosynthesis. Plants were entrained to a 12-hour-day:12-hour-night light regimen and then monitored or sampled during a subsequent period of constant light. We observed circadian oscillations in ribulose-1,5-bisphosphate (RuBP) levels, and to a lesser extent in phosphoglyceric acid (PGA) levels, that closely paralleled oscillations in photosynthesis. However, the enzyme activity and activation state of the enzyme responsible for the conversion of RuBP to PGA, ribulose-1,5-bisphosphate carboxylase/oxygenase, showed no discernible circadian oscillation. Hence, we examined the possibility of circadian effects on RuBP regeneration. Neither ribulose-5-phosphate kinase activity nor the level of ATP fluctuated in constant light. Oscillations in triose-phosphate levels were out of phase with those observed for RuBP and PGA.