Human recombinant butyrylcholinesterase purified from the milk of transgenic goats interacts with beta-amyloid fibrils and suppresses their formation in vitro.

BACKGROUND: In Alzheimer's disease (AD), brain butyrylcholinesterase (BChE) co-localizes with beta-amyloid (Abeta) fibrils. AIMS: In vitro testing of the significance of this phenomenon to AD progress. METHODS: A thioflavine T (ThT) fluorogenic assay, photo-induced cross-linking and quantifiable electron microscopy served to compare the effect on Abeta fibril formation induced by highly purified recombinant human BChE (rBChE) produced in the milk of transgenic goats with that of serum-derived human BChE. RESULTS: Both proteins at 1:50 and 1:25 ratios to Abeta dose-dependently prolonged the ThT lag time and reduced the apparent rate of Abeta fibril formation compared to Abeta alone. Photo-induced cross-linking tests showed that rBChE prolonged the persistence of amyloid dimers, trimers and tetramers in solution, whereas Abeta alone facilitated precipitation of such multimers from solution. Transmission electron microscopy showed that rBChE at 1:100 to Abeta prevented the formation of larger, over 150-nm-long, Abeta fibrils and reduced fibril branching compared to Abeta alone as quantified by macro programming of Image Pro Plus software. CONCLUSION: Our findings demonstrate that rBChE interacts with Abeta fibrils and can attenuate their formation, extension and branching, suggesting further tests of rBChE, with unlimited supply and no associated health risks, as a therapeutic agent for delaying the formation of amyloid toxic oligomers in AD patients.

Acetylcholinesterase/C terminal binding protein interactions modify Ikaros functions, causing T lymphopenia.

Hematological changes induced by various stress stimuli are accompanied by replacement of the primary acetylcholinesterase (AChE) 3' splice variant acetylcholinesterase-S (AChE-S) with the myelopoietic acetylcholinesterase-R (AChE-R) variant. To search for putative acetylcholinesterase-S interactions with hematopoietic pathways, we employed a yeast two-hybrid screen. The transcriptional co-repressor C-terminal binding protein (CtBP) was identified as a protein partner of the AChE-S C terminus. In erythroleukemic K562 cells, AChE-S displayed nuclear colocalization and physical interaction with CtBP. Furthermore, co-transfected AChE-S reduced the co-repressive effect of CtBP over the hematopoietic transcription factor, Ikaros. In transgenic mice, overexpressed human (h) AChE-S mRNA induced selective bone marrow upregulation of Ikaros while suppressing FOG, another transcriptional partner of CtBP. Transgenic bone marrow cells showed a correspondingly elevated potential for producing progenitor colonies, compared with controls, while peripheral blood showed increased erythrocyte counts as opposed to reduced platelets, granulocytes and T lymphocytes. AChE's 3' alternative splicing, and the corresponding changes in AChE-S/CtBP interactions, thus emerge as being actively involved in controlling hematopoiesis and the potential for modulating immune functions, supporting reports on malfunctioning immune reactions under impaired splice site selection.

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses.

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

Intracardiac echogenic focus: no apparent association with structural cardiac abnormality.

OBJECTIVE: The aim of our study was to evaluate whether intracardiac echogenic foci (ICEFs) may be associated with increased risk for structural cardiac anomalies in the low-risk population. METHODS: During a 24-month period, 3,744 low-risk patients were prospectively screened for ICEFs by prenatal sonography. The study group was composed of 138 fetuses (3.7%) with ICEF. The control group was composed of 167 fetuses without ICEF. In all fetuses a complete echocardiographic evaluation was performed. RESULTS: Among the 138 fetuses in the study group, 108 (78%) ICEFs were found in the left ventricle, 25 (18%) were found in the right ventricle, and 5 (4%) were found to be bilateral. No statistically significant difference was found between the study and the control group regarding the presence of cardiac anomalies. Only 1 case (0.7%) of pulmonic stenosis was found in the study group, compared to 1 case (0.6%) of bicuspid aortic valve in the control group. CONCLUSIONS: We conclude that ICEFs found in low-risk patients are not associated with a significant increase in the risk of cardiac anomalies.

Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery.

Classic in vitro studies show that the Hsp70 chaperone system from Escherichia coli (DnaK-DnaJ-GrpE, the DnaK system) can bind to proteins, prevent aggregation, and promote the correct refolding of chaperone-bound polypeptides into native proteins. However, little is known about how the DnaK system handles proteins that have already aggregated. In this study, glucose-6-phosphate dehydrogenase was used as a model system to generate stable populations of protein aggregates comprising controlled ranges of particle sizes. The DnaK system recognized the glucose-6-phosphate dehydrogenase aggregates as authentic substrates and specifically solubilized and refolded the protein into a native enzyme. The efficiency of disaggregation by the DnaK system was high with small aggregates, but the efficiency decreased as the size of the aggregates increased. High folding efficiency was restored by either excess DnaK or substoichiometric amounts of the chaperone ClpB. We suggest a mechanism whereby the DnaK system can readily solubilize small aggregates and refold them into active proteins. With large aggregates, however, the binding sites for the DnaK system had to be dynamically exposed with excess DnaK or the catalytic action of ClpB and ATP. Disaggregation by the DnaK machinery in the cell can solubilize early aggregates that formed accidentally during chaperone-assisted protein folding or that escaped the protection of "holding" chaperones during stress.

Shwachman--Diamond syndrome associated with autoimmune phenomena.

A 2-year-old boy was evaluated for failure to thrive, hypotonia and developmental delay. The child exhibited all the criteria of Shwachman-Diamond syndrome, i.e., short stature, metaphyseal dysostosis, pancreatic insufficiency and neutropenia. Liver function tests were abnormal. Marked edema together with pericardial effusion appeared during the period of follow-up. Hypothyroidism attributed to autoimmune thyroiditis was diagnosed, and other autoantibodies were detected as well. We suggest that an autoimmune baseline profile and follow-up should be part of the work-up and management of patients with Shwachman-Diamond syndrome. Moreover, the finding of autoantibodies might offer a new insight towards understanding the pathogenesis of this condition.

Temperature-controlled activity of DnaK-DnaJ-GrpE chaperones: protein-folding arrest and recovery during and after heat shock depends on the substrate protein and the GrpE concentration.

Heat-shock proteins DnaK, DnaJ, and GrpE (KJE) from Escherichia coli constitute a three-component chaperone system that prevents aggregation of denatured proteins and assists the refolding of proteins in an ATP-dependent manner. We found that the rate of KJE-mediated refolding of heat- and chemically denatured proteins is decreased at high temperatures. The efficiency and reversibility of protein-folding arrest during and after heat shock depended on the stability of the complex between KJE and the denatured proteins. Whereas a thermostable protein was released and partially refolded during heat shock, a thermolabile protein remained bound to the chaperone. The apparent affinity of GrpE and DnaJ for DnaK was decreased at high temperatures, thereby decreasing futile consumption of ATP during folding arrest. The coupling of ATP hydrolysis and protein folding was restored after the stress. This strongly indicates that KJE chaperones are heat-regulated heat-shock proteins which can specifically arrest the folding of aggregation-prone proteins during stress and preferentially resume refolding under conditions that allow individual proteins to reach and maintain a stable native conformation.

The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network.

The role of small heat-shock proteins in Escherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins.

GroES binding regulates GroEL chaperonin activity under heat shock.

Chaperonins GroEL14 and GroES7 are heat-shock proteins implicated in the molecular response to stress. Protein fluorescence, crosslinking and kinetic analysis revealed that the bond between the two otherwise thermoresistant oligomers is regulated by temperature. As temperature increased, the affinity of GroES7 and the release of bound proteins from the chaperonin concomitantly decreased. After heat shock, GroES7 rebinding to GroEL14 and GroEL14GroES7 particles correlated with the restoration of optimal protein folding/release activity. Chaperonins thus behave as a molecular thermometer which can inhibit the release of aggregation-prone proteins during heat shock and restore protein folding and release after heat shock.

A one-way valved atrial septal patch: a new surgical technique and its clinical application.

Patients who undergo surgical repair of congenital heart defects, characterized by a hypoplastic right ventricle or high pulmonary vascular resistance, are at high risk for the development of postoperative right heart failure. This risk may discourage the surgical team from carrying out a biventricular or complete repair in such patients. To reduce the risk for right heart failure, we developed a one-way, valved, atrial septal patch to serve as an artificial one-way foramen ovale and tested it in an animal model. By permitting right-to-left shunt, this device decompresses the failing right ventricle and maintains systemic cardiac output. The device has been used in 15 patients divided into three different groups: group 1 (n = 8), patients with a hypoplastic right ventricle and pulmonic stenosis or atresia, seven of whom underwent a biventricular repair; group 2 (n = 5), patients with evidence of pulmonary disease after longstanding left-to-right shunt caused by a correctable atrial or ventricular septal defect, all of whom had a complete repair; group 3, two patients with acute right heart failure in whom the device was used as a last option of treatment to wean them from cardiopulmonary bypass. This article presents our data in regard to the use of the one-way, valved, atrial septal patch and the indications for its clinical use.

The protein-folding activity of chaperonins correlates with the symmetric GroEL14(GroES7)2 heterooligomer.

Chaperonins GroEL and GroES form, in the presence of ATP, two types of heterooligomers in solution: an asymmetric GroEL14GroES7 "bullet"-shaped particle and a symmetric GroEL14(GroES7)2 "football"-shaped particle. Under limiting concentrations of ATP or GroES, excess ADP, or in the presence of 5'-adenylyl imidodiphosphate, a correlation is seen between protein folding and the amount of symmetric GroEL14(GroES7)2 particles in a chaperonin solution, as detected by electron microscopy or by chemical crosslinking. Kinetic analysis suggests that protein folding is more efficient when carried out by a chaperonin solution populated with a majority of symmetric GroEL14(GroES7)2 particles than by a majority of asymmetric GroEL14GroES7 particles. The symmetric heterooligomer behaves as a highly efficient intermediate of the chaperonin protein folding cycle in vitro.

Increased efficiency of GroE-assisted protein folding by manganese ions.

This study addresses the role of ATP-bound and free Mg2+ and Mn2+ ions in the activation and modulation of chaperonin-assisted refolding of urea-denatured malate dehydrogenase. As compared with Mg2+, Mn2+ ions caused a significant increase in the rate of GroE-assisted malate dehydrogenase refolding and, concomitantly, a decrease in the rate of ATP hydrolysis. Moreover, Mn2+ increases the affinity of GroES for GroEL, even in the presence of saturating amounts of Mg2+. Chemical cross-linking showed that lower concentrations of Mn-ATP as compared with Mg-ATP are needed to form both asymmetric GroEL14GroES7 and symmetric GroEL14(GroES7)2 particles. The manganese-dependent increase in the rate of protein folding concurred with a specific increase in the amount of symmetric GroEL14-(GroES7)2 particles detected in a chaperonin solution. Thus, Mn2+ is a cofactor that can markedly increase the efficiency of the chaperonin reaction in vitro. Mn2+ ions can serve as an important tool for analyzing the molecular mechanism and the structure of chaperonins.

Effect of free and ATP-bound magnesium and manganese ions on the ATPase activity of chaperonin GroEL14.

Hydrolysis of ATP by the GroEL14 chaperonin oligomer is activated and modulated by Mg2+ or Mn2+ ions. Mg-ATP and Mn-ATP can serve as substrates of the reaction and bind in a positively cooperative manner to the same catalytic sites on GroEL14, with similar binding constants in the micromolar range. In addition, millimolar amounts of Mg2+ and Mn2+ ions can further activate the GroEL14-ATPase while interacting with low-affinity noncatalytic sites on the chaperonin. The extent of ATPase activation by Mn2+ is half of that by Mg2+ ions. When both Mg2+ and Mn2+ ions are present in the same reaction, Mn2+ behaves as a noncompetitive partial inhibitor of the Mg-dependent ATPase. This inhibition requires the presence of ADP in the catalytic site. The binding affinity of Mn-ADP to the site is significantly higher than that of Mg-ADP. A slower release of Mn-ADP from the catalytic site thus changes the rate-determining step of the GroEL14-ATPase cycle. In the cell, the concentrations of Mg2+ and Mn2+ ions are such that both divalent ions may modulate chaperonin activity.

A one-way, valved, atrial septal patch in the management of postoperative right heart failure. An animal study.

Patients who undergo surgical repair of congenital heart defects that are characterized by hypoplastic right ventricle or pulmonary hypertension are at high risk for the development of postoperative right heart failure. To minimize this risk, a new one-way, valved, atrial septal patch was developed that serves as an artificial one-way foramen ovale. The feasibility of this device was tested in five dogs in which reversible right heart failure had been induced. Use of the one-way valved patch significantly improved right ventricular performance and reduced right ventricular hydrostatic pressures. The interatrial pressure gradient was reduced from 10 +/- 3.5 mm Hg during right heart failure to 4.4 +/- 1.4 mm Hg. When the device was opened, cardiac output increased significantly. This hemodynamic improvement was achieved at the expense of systemic arterial desaturation, which was, however, well tolerated. When the state of right heart failure was reversed, the one-way valved patch spontaneously closed, allowing no interarterial shunting. The results of this experimental trial suggest that this device can play a useful role in selected patients in whom postoperative right heart failure can be anticipated after surgical repair of certain congenital heart defects.

Effect of divalent cations on the molecular structure of the GroEL oligomer.

Structural analysis, by chemical cross-linking with glutardialdehyde (GA), and by urea denaturation, was carried out for the chaperonin oligomer GroEL14 from Escherichia coli. The cross-linking reaction of GroEL14 presents two phases: a rapid intralayer cross-linking reaction, which first occurs between the monomers of individual GroEL7 heptameric rings, and a slow interlayer cross-linking reaction, which later occurs between the two stacked heptameric rings of the GroEL14 oligomer. The biphasic behavior of the cross-linking reaction indicates that the surfaces of contact between GroEL monomers within individual heptameric rings are more extensive than the surfaces of contact between the two GroEL7 rings of the oligomer. Millimolar amounts of the divalent cations Mg2+, Mn2+, Ca2+, or Zn2+, but not of monovalent ions, increase the velocity of both intra- and interlayer cross linking. Divalent cations increase the stability of the native GroEL14 oligomer in urea. In contrast, Mg2+ activates ATP hydrolysis by GroEL14, with an activation constant in the micromolar range, while Ca2+ does not significantly assist ATP hydrolysis. It is concluded that divalent cations affect the structure of GroEL14 in particular the contacts between monomers within the GroEL7 heptameric layers. The effect of divalent cations on the structure of the chaperonin molecule is quantitatively and qualitatively distinct from that of magnesium ions on the chaperonin ATPase activity.

Imidazoline binding sites in human placenta: evidence for heterogeneity and a search for physiological function.

1. An alpha 2-adrenoceptor antagonist, idazoxan, that binds to both alpha 2-adrenoceptors and to imidazoline sites (IR), has been used to characterize human placental IR. Human placenta is shown to be the richest source of IR (1800 +/- 100 fmol mg-1 protein; Kd 38.9 +/- 3.4 nM). 2. Primary cells derived from human placenta and grown in monolayers, also displayed a high density of receptors (3209 +/- 136 fmol mg-1 in cytotrophoblasts and 3642 +/- 144 fmol mg-1 protein in syncytiotrophoblast enriched cell culture). 3. [3H]-idazoxan did not show binding characteristics of alpha 2-adrenoceptors in human placental membranes or human trophoblastic cells, thus making it a ligand of choice to study the imidazoline site. The tissue appeared to be lacking alpha 2-adrenoceptors in that other alpha 2-adrenoceptor ligands, [3H]-rauwolscine and [3H]-clonidine, do not bind to alpha 2-adrenoceptors in human placenta. 4. IRs are localized on the cell surface, as determined by the release of bound [3H]-idazoxan from cells, when washed with high ionic/acidic medium. 5. Imidazoline receptors of human placenta display high affinity for amiloride (72 +/- 27 nM). The high affinity was used as a criterion to classify IR to IRa subtype (placenta, rabbit kidney, rabbit liver and rabbit adipose cells) as opposed to the IRb subtype which display low affinity for amiloride (greater than 2 microM, in all the other tissues).6. Several novel ligands comprising a guanido functional group attached to an aromatic residue (e.g. benziliden-amino-guanidine (BAG), guanido pyrole) display pronounced selectivity for IR over the M2-adrenoceptors as the affinity of BAG is about 40 fold higher (Kd= 18.9 +/- 13.8 nM in human placenta), than the affinity for M2-adrenoceptors (Kd = 768 +/- 299 nM in human platelets). Imidazoline sites bind selectively BAG and other guanido ligands thus indicating a distinct structural requirement at its site of binding.7. K+ channel blockers and monovalent ions (e.g. Cs' and NH4+) interfere with idazoxan binding to IR, indicating a possible involvement of IR in K+ transport.

Is imidazoline site a unique receptor? A correlation with clonidine-displacing substance activity.

Many specific hypotensive drugs acting via the central alpha 2-adrenoceptors were designed based on their imidazoline/guanidine structure for use as antihypertensives. This unique structure, which is missing in the alpha 2-adrenoceptor natural ligands, led to the search for an endogenous, nonadrenergic ligand, and later on, for its putative receptor. Indeed, an endogenous ligand designated the "clonidine displacing substance" (CDS), was isolated and purified from bovine brain, and characterized in various cells. The most intriguing feature of CDS is its hypertensive action upon injection into the rostral ventrolateral medulla and its competition with clonidine. Is CDS a natural agonist which is displaced by clonidine or other hypotensive drugs? Does the unique imidazoline/guanidine structure imply a unique recognition site? Recent studies reported that an imidazoline site, distinct from the alpha 2-adrenoceptor, is abundant in many tissues, and it preferentially recognizes the imidazolino-guanidino type ligands. The physiological role of these sites is still not well defined. In the present study we show that the richest tissue in imidazoline sites is human placenta (1800 +/- 100 fmol/mg protein). The sites are distributed on the cell surface, as observed in studies of binding to intact cytotrophoblasts and cultured trophoblasts originating from human placenta. Binding studies show that the imidazoline site displays a unique pharmacological profile distinct from the alpha 2-adrenoceptor (eg, benzylidenamino-guanidine, Ki = 18.9 +/- 13.8 nmol/L for the imidazoline sites and Ki = 768 +/- 299 nmol/L for the alpha 2-adrenoceptors; guanidopyrol, Ki = 11.2 +/- 6.3 nmol/L for imidazoline sites and Ki = 10100 +/- 1515 nmol/L for the alpha 2-adrenoceptors).(ABSTRACT TRUNCATED AT 250 WORDS)

Bloom's syndrome in an Iranian Jewish male.

Bloom's syndrome is described in an Iranian Jewish male who subsequently developed myocardial disease. This may represent the first definitely non Ashkenazi Jewish patient in the literature and the only one to develop this complication.

Imidazoline receptors in rat liver cells: a novel receptor or a subtype of alpha 2-adrenoceptors?

An imidazoline/guanidine receptor has been characterized in rat liver cells. Binding of [3H]idazoxan, a selective benzodioxan antagonist, to imidazoline receptor on intact fresh hepatocytes (Bmax = 801 +/- 23 fmol/mg protein, Kd = 11 +/- 0.8 nM) and to liver membranes (Bmax = 400 +/- 38 fmol/mg protein, Kd = 10 +/- 2 nM) was saturable at 4 degrees C within 3.5 h and at 30 degrees C within 30 min, respectively. Rat lung membranes had more imidazoline sites (Bmax = 578 +/- 30 fmol/mg protein, Kd = 14 +/- 1.4 nM) than alpha 2-adrenoceptors (Bmax = 175.0 +/- 20.0 fmol/mg protein, Kd = 4.8 +/- 2.0 nM). We also screened other tissues for imidazoline sites; the ratio of adrenoceptors to total sites labeled with [3H]idazoxan displaced by cirazoline was lower in rat lung compared to rat brain and human platelets. The imidazoline receptor has common pharmacological properties with alpha 2-adrenoceptors, although it is not a subtype of the adrenoceptor, since it bound neither the endogenous agonists norepinephrine and epinephrine, nor the selective alpha 2-antagonists yohimbine and phentolamine. All guanidine type alpha 2-adrenoceptor drugs (e.g. guanbenz, guanoxan) and imidazolines (e.g., UK-14,304, naphazoline) competed with high affinity for the liver imidazoline receptor. The lack of effect by Gpp(NH)p, a non-hydrolysable GTP analogue, on the affinity of guanidine- and imidazoline-type ligands for liver imidazoline receptors suggests that the mode of action of these drugs at imidazoline receptors is different than at conventional alpha 2-adrenoceptors. Ionic changes were considered as a possible mechanism underlying the alpha 2-adrenoceptor effects in various cells. Opening of K+ channels by alpha 2-adrenoceptors agonists is a pathway which might be shared by imidazoline-type agonists at imidazoline sites. Indeed, 4-aminopyridine, a K+ channel blocker, inhibited the specific binding of [3H]idazoxan to liver cells with an IC50 of 0.34 +/- 0.07 mM a concentration which is effective in blocking K+ channels in neuronal cells. Similarly, Cs+ and NH4+ effectively interfered with [3H]idazoxan binding, suggesting a possible coupling of imidazoline sites to K+ gating. The endogenous ligand clonidine-displacing substance (CDS), which was isolated from bovine brain and which binds to alpha 2-adrenoceptors in brain membranes and human platelets competed with idazoxan at rat liver imidazoline receptors.(ABSTRACT TRUNCATED AT 400 WORDS)