Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.     

Localization of a highly immunogenic region on the acetylcholine receptor alpha-subunit

Antibodies to synthetic peptides were employed in order to map domains on the alpha-subunit of the acetylcholine receptor to which several monoclonal antibodies are directed. Five peptides corresponding to residues 1-20, 126-143, 169-181, 330-340 and 351-368 of the receptor alpha-subunit were synthesized and antibodies against them were elicited. The anti-peptide antibodies were employed along with the monoclonal antibodies to identify fragments of S. aureus V8 protease digested- alpha-subunit in immunoblotting experiments. Our results demonstrate that a highly immunogenic region of the alpha-subunit is located on a carboxy-terminal 14 kDa portion of the alpha-subunit. This region also seems to undergo antigenic changes during muscle development. A monoclonal antibody directed against the cholinergic binding site of the acetylcholine receptor reacted with an 18 kDa segment of the alpha-subunit which bound alpha-bungarotoxin as well as antibodies directed against peptide 169-181.     

Interaction between inclusions embedded in membranes.

We calculate the membrane-induced interaction between inclusions, in terms of the membrane stretching and bending moduli and the spontaneous curvature. We find that the membrane-induced interaction between inclusions varies nonmonotonically as a function of the inclusion spacing. The location of the energy minimum depends on the spontaneous curvature and the membrane perturbation decay length, where the latter is set by the membrane moduli. The membrane perturbation energy increases with the inclusion radius. The Ornstein-Zernike theory, with the Percus-Yevick closure, is used to calculate the radial distribution function of inclusions. We find that when the spontaneous curvature is zero, the interaction between inclusions due to the membrane deformation is qualitatively similar to the hard-core interaction. However, in the case of finite spontaneous curvature, the effective interaction is dramatically modified.     

Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.     

Polymer-induced membrane contraction, phase separation, and fusion via Marangoni flow.

Experiments have shown that the depletion of polymer in the region between two apposed (contacting or nearly contacting) bilayer membranes leads to fusion. In this paper we show theoretically that the addition of nonadsorbing polymer in solution can promote lateral contraction and phase separation of the lipids in the outer monolayers of the membranes exposed to the polymer solution, i.e., outside the contact zone. This initial phase coexistence of higher- and lower-density lipid domains in the outer monolayer results in surface tension gradients in the outer monolayer. Initially, the inner layer lipids are not exposed to the polymer solution and remain in their original "unstressed" state. The differential stresses on the bilayers give rise to a Marangoni flow of lipid from the outer monolayers in the "contact zone" (where there is little polymer and hence a uniform phase) to the outer monolayers in the "reservoir" (where initially the surface tension gradients are large due to the polymer-induced phase separation). As a result, the low-density domains of the outer monolayers in the contact zone expose their hydrophobic chains, and those of the inner monolayers, to the solvent and to each other across the narrow water gap, allowing fusion to occur via a hydrophobic interaction. More generally, this type of mechanism suggests that fusion and other intermembrane interactions may be triggered by Marangoni flows induced by surface tension gradients that provide "action at a distance" far from the fusion or interaction zone.     

The results after transrectal prostate biopsy with 12 biopsy cores taken

This report describes the clinical value of transrectal prostate biopsy during which 12 biopsy cores are taken in comparison to the classical sextant method. There were 106 patients included in the study, who had transrectal prostate biopsy (TRB) due to abnormal finding after digitorectal examination (DRE) and/or values of PSA > 4 ng/ml in the period from 4 October 2001 till 14 August 2002. There were 117 biopsies with 12 biopsy cores taken, 6 cores from each lobe. Prostate cancer was confirmed in 49 patients (46%). Out of total number of confirmed cancer cases, initial biopsy detected 94%. There were three patients who had suspicious DRE finding, with PSA value of < 4 ng/ml, but cancer was not detected in any of them. In the patient group with PSA value between 4-10 ng/ml, cancer was detected in 26% of them and in the group with PSA value > 10 ng/ml cancer was detected in 58%. The most common Gleason score in the case of cancer was 7 (43%). During the biopsy procedure, 3 patients experienced strong vasovagal reactions, meaning that out of 117 biopsies incidence of complications was 2.6%. Few days after the biopsy, two patients developed urogenital tract infections (1.7%) and right after the procedure, there was one case of strong hematuria (0.8%) and strong rectal bleeding (0,8%) that needed hospitalization. Our results regarding the incidence of complications do not differ much from the results in the literature. According to data in the literature regarding sextant biopsy, 15-34% of cancer cases remain undiagnosed at initial biopsy. The method of 12 biopsy cores fails to diagnose only 6% of all cancers, but it is important to note that in the mentioned period, re-biopsy was indicated only in 11 from 60 patients with negative biopsies.     

The chemical composition of the meat of fat dormice (<Emphasis Type="Italic">Glis glis</Emphasis> L.)

The fat dormouse (Glis glis) is a traditional game species in the Republic of Croatia. Although today the fat dormouse is not frequently caught as game, it is still a source of animal protein in human nutrition in certain rural areas of Croatia. In this paper the chemical analysis of fat dormouse meat is presented. The average values for the quantity of water, fat, protein and ash in dormouse meat are comparable with the chemical composition of the meat of rabbits and brown hares, except for the important fact that rabbit and hare meat contains a greater quantity of fat on average. According to our results, the meat of fat dormice can be categorised as dietary food, characterised by a small percentage of fat (mean 2.83%) and a high amount of protein (mean 21.01%).     

Comparative morphology of incisor enamel and dentin in humans and fat dormice (Glis glis)

The structure of teeth in all living beings is genetically predetermined, although it can change under external physiological and pathological factors. The author's hypothesis was to indicate evolutional shifts resulting from genetic, functional and other differences. A comparative study about certain characteristics of incisors in humans and myomorpha, the fat dormouse (Glis glis) being their representative as well, comprised measurements of enamel and dentin thickness in individual incisor segments, evaluation of external enamel index, and also assessment of histological structure of enamel and dentin. The study results involving dormice showed the enamel to be thicker in lower than in the upper teeth, quite contrary to enamel thickness in humans. In the upper incisors in dormice the enamel is the thickest in the medial layer of the crown, and in the cervical portion of the crown in the lower incisors. The thickness of dentin in dormice is greater in the oral than in the vestibular side. These findings significantly differ from those reported in reference literature, but they are based on the function of teeth in dormice. Histological characteristics of hard dental tissues in dormice are similar to those in humans, with exception of uniserial structure of enamel and appearance of dentinoenamel junction.     

ErbB2 degradation mediated by the co-chaperone protein CHIP

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.