Mapping the extracellular topography of the α-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain

The extracellular surface of theα-chain ofTorpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR. was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptidesα1–16,α89–104,α158–174,α262–276, andα388–408. Lower, but significant, levels of reactivity were obtained with antibodies against peptidesα67–82,α78–93,α100–115, andα111–126. On the other hand, free AChR bound high levels of antibodies against peptidesα34–49,α78–93,α134–150,α170–186, andα194–210. It also bound moderate levels of antibodies against peptidesα262–276 andα388–408. Low, yet significant, levels of binding were exhibited by antibodies against peptidesα45–60,α111–126, andα122–138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Deletion polymorphism of the angiotensin I-converting enzyme gene in elderly patients with coronary heart disease

Controversy exists as to whether the deletion/deletion (DD) genotype of angiotensin l-converting enzyme (ACE) gene polymorphism is associated with coronary heart disease (CHD). There are only a few studies dealing with this issue in the elderly, also with controversial results. The aim of this study was the assessment of correlation between genetic markers and the risk of CHD in the elderly. The results indicated DD genotype importance for CHD in the elderly as proven by discriminant analysis (chi2 = 25.77; df = 16; p = 0.0620). However, the use of univariate method demonstrated no correlation between DD genotype of ACE gene polymorphism and coronary artery disease. D allele of ACE gene was associated with higher activities of ACE plasma. A weak, but increased risk of MI is associated with high frequency of DD genotype in the elderly. Strong correlation between ACE polymorphism and ACE plasma activities was demonstrated.     

Cell apoptosis as assessed by M30 expression in keratoacanthoma and squamous cell carcinoma

Involution displayed by keratoacanthoma (KA) represents an important difference between KA and squamous cell carcinoma (SCC). It has been suggested that apoptosis plays a part in process of involution of KA. Altogether 150 specimens were included in this study, 30 cases of each; normal skin (NS), proliferative (pKA) and regressing keratoacanthoma (rKA), well differentiated (wdSCC) and poorly differentiated (pdSCC) squamous cell carcinoma. All samples were examined immunohistochemically for expression of M30 protein. A significantly lower number of M30 positive cells has been detected in NS as compared to skin tumors examined (p<0.001), except for rKA (p=0.057). The highest percentage of M30 positive cells was detected in pdSCC (p<0.001) as compared with all other examined groups. Keratinocytes of normal and changed epidermis expressing higher levels of M30 protein were predominately found in sun-exposed areas (chi2=14.93; p=0.060). There was an increasing trend of M30 protein expression with increasing age of the patient in NS and skin tumors examined. Majority of skin tumors with higher percentage of M30 positive cells tended to display higher Ki-67 expression. M30 expression was highly correlated with bak (r=0.811; p=0.048) and granzyme B expression in rKA (r=0.733; p=0.015). Cell apoptosis as assessed by M30 expression is, generally, increased in examined skin tumors and related to cell proliferation. Cell apoptosis mediated by bak and granzyme B expression could contribute to KA regression.     

Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.     

Peroxisome proliferator-activated receptor-gamma1 is dephosphorylated and degraded during BAY 11-7085-induced synovial fibroblast apoptosis

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-gamma has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-gamma agonist 15d-PGJ2; the expression of PPAR-gamma in these cells was studied. Both PPAR-gamma1 and PPAR-gamma2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-gamma1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-gamma1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-gamma1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-gamma1 protein degradation. PPAR-gamma1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-gamma1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-gamma1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-gamma dephosphorylation and degradation. Furthermore, PPAR-gamma antagonist BADGE induced PPAR-gamma1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-gamma1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-gamma1 dephosphorylation but before PPAR-gamma1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts.     

Reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells

Our recent studies have shown that the de novo sphingolipids play a role in apoptosis of photosensitized cells. To elucidate the involvement of the de novo sphingolipids in reactive oxygen species (ROS) production and mitochondrial depolarization during apoptosis, the stress inducer photodynamic therapy (PDT) with the photosensitizer Pc 4 was used. In Jurkat cells PDT-triggered ROS production or mitochondrial membrane potential (deltapsi(m)) loss was not prevented by the de novo sphingolipid synthesis inhibitor ISP-1. However, PDT + C16-ceramide led to enhanced mitochondrial depolarization and DEVDase activation. The superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected Jurkat cells from ROS generation and apoptosis, but not from deltapsi(m) reduction. Sphinganine or C16-ceramide counteracted MnTBAP-induced protection from apoptosis in Jurkat, as well as CHO cells. In LY-B cells, CHO-derived mutants deficient in serine palmitoyltransferase (SPT) activity and the de novo sphingolipid synthesis, mitochondrial depolarization, but not ROS generation, was suppressed post-PDT. In LY-B cells transfected with the SPT component LCB1, deltapsi(m) collapse post-PDT was restored. The data support the following hypotheses: MnTBAP protects against apoptosis via steps downstream of deltapsi(m) loss; de novo sphingolipids are not required for ROS generation, but can play a role in deltapsi(m) dissipation in photosensitized apoptotic cells.     

15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.     

Circadian components in energy and tension and their relation to physiological activation and performance

The purpose of the study was to examine validity of R. Thayer's activation model regarding 24h variations of two subjective dimensions of activation (Energy and Tension), and their 24 h relations with indices of physiological activation and performance efficiency. The participants of the study (n = 28 females) spent 26 h under controlled laboratory conditions. Self-ratings of subjective activation and measurements of oral temperature, electrodermal activity, and performance on a visual vigilance task were done every 4 h. Twenty-four-hour variations were examined by means of repeated measures analyses of variance and by group mean cosinor analyses before and after controlling for the data trends. Self-ratings on both dimensions of subjective activation showed significant 24 h variation. Energy showed both nonrhythmic and endogenously determined circadian variation, while 24h variation of tension was dominantly nonrhythmic and most probably determined by exogenous factors. Significant 24 h covariation was found between energy and body temperature. A negative correlation between 24 h variation of energy and tension was also found. Considering low and intermediate levels of subjective activation established over the 24 h in this study, the association of the two dimensions of subjective activation did not prove to be consistent with the assumptions of Thayer's model.