The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings.     

Dominant negative suppression of arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor

We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes.     

Interaction of cryptochrome 1, phytochrome, and ion fluxes in blue-light-induced shrinking of Arabidopsis hypocotyl protoplasts

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl- and K+. The postshrinking volume recovery is achieved by K+ and Cl- influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.     

Constitutive Alzheimer's-type tau epitopes in a neuritogenic rat CNS cell line

Paired helical filaments (PHFs) of Alzheimer's disease (AD) largely comprise hyperphosphorylated forms of the cytoskeletal protein tau. AD-type tau phosphoepitopes, detected by various monoclonal antibodies, are absent from normal adult neurons, but recent studies have shown that their expression may contribute to neuritogenesis and axon differentiation in the developing nervous system. Therefore, we have examined a brain nerve cell line that is spontaneously neuritogenic for possible expression of AD-type tau epitopes. The neuritogenic rat brain cell line B103 was found to constitutively produce two-AD related epitopes of tau, detected by cellular immunofluorescence studies with the PHF-1 and Alz-50 monoclonal antibodies. Biochemical studies showed that the antibodies bound to proteins within the molecular, weight range expected for phosphorylated tau isoforms. Further verification was established by use of tau antisense oligomers, which eliminated cellular immunofluorescence due to the AD-related monoclonals and polyclonal anti-tau but did not eliminate fluorescence due to anti-tubulin. Cells treated with tau antisense were not neurite-free. Neurites that remained, however, were abnormal, generally short and wavy in appearance. Cellular distribution of the tau epitopes was found to be particularly interesting. Alz-50 recognized only cytoplasmic tau whereas PHF-1 recognized nuclear tau as well as cytoplasmic. Thus, the two epitopes, are morphologically segregated within the cell. Because subcellular segregation of tau is compromised in Alzheimer's disease, mechanisms that segregate AD-type phosphotau epitopes in B103 cells may have relevance to this neurodegenerative disorder.     

Soluble proteins modified with acetaldehyde and malondialdehyde are immunogenic in the absence of adjuvant

Recent studies have shown that the alcohol metabolites malondialdehyde and acetaldehyde can combine to form a stable adduct (MAA) on proteins. This adduct has been detected in the livers of rats chronically consuming ethanol, and serum antibodies to MAA have been observed at significantly higher concentrations in ethanol-fed when compared with pair-fed or chow-fed control rats. More recently, preliminary studies have strongly suggested that the MAA adduct is capable of stimulating antibody responses to soluble proteins in the absence of adjuvants. The antibodies produced recognize either the MAA epitope or the carrier protein itself. Therefore, it was the purpose of this study to examine the potential immunogenicity of MAA-modified exogenous proteins in the absence of adjuvants. Balb/c mice were immunized in the presence or absence of adjuvant with different concentrations of unmodified or MAA-modified proteins. The antibody response to both the MAA epitope and unmodified protein epitopes were determined by ELISA. In the absence of adjuvant, significant antibody responses were induced to both the MAA epitope and nonmodified protein epitopes. Smaller immunizing doses of MAA-protein conjugate favored the production of antibodies to nonmodified proteins, whereas larger doses induced a strong anti-MAA response. In studies to begin determining a mechanism for the specificity of the response in the absence of adjuvants, peritoneal macrophages were found to bind and degrade MAA-adducted proteins through the use of a scavenger receptor. This indicated that MAA-adducted proteins may be specifically taken up and epitopes presented to the humoral immune system in the absence of adjuvants. Importantly, these are the first data showing that an alcohol-related metabolite can induce an antibody response in the absence of adjuvant and suggesting a mechanism by which antibody to the MAA adduct or its carrier (exogenous or endogenous) proteins may be generated in vivo.     

Multiple sclerosis: in situ evidence for antibody- and complement-mediated demyelination

Blue light responses in higher plants can be mediated not only by specific blue light receptors, but also by the red/far-red photoreversible phytochrome system. The question of interdependence between these photoreceptors has been debated over many years. The availability of Arabidopsis mutants for the blue light receptor CRY1 and for the two major phytochromes phyA and phyB allows a reinvestigation of this question. The analysis of photocontrol of seed germination, inhibition of hypocotyl growth and anthocyanin accumulation clearly demonstrates that (i) phyA shows a strong control in blue light responses especially at low fluence rates; (ii) phyB mediated induction reactions can be reversed by subsequent blue light irradiations; and (iii) CRY1 mediates blue light controlled inhibition of hypocotyl growth only at fluence rates higher than 5 mumol m-2s-1 and independently of phytochrome A and B.     

Phytochrome A regulates red-light induction of phototropic enhancement in Arabidopsis

Phytochrome A (phyA) and phytochrome B photoreceptors have distinct roles in the regulation of plant growth and development. Studies using specific photomorphogenic mutants and transgenic plants overexpressing phytochrome have supported an evolving picture in which phyA and phytochrome B are responsive to continuous far-red and red light, respectively. Photomorphogenic mutants of Arabidopsis thaliana that had been selected for their inability to respond to continuous irradiance conditions were tested for their ability to carry out red-light-induced enhancement of phototropism, which is an inductive phytochrome response. We conclude that phyA is the primary photoreceptor regulating this response and provide evidence suggesting that a common regulatory domain in the phyA polypeptide functions for both high-irradiance and inductive phytochrome responses.     

Modified phage peptide libraries as a tool to study specificity of phosphorylation and recognition of tyrosine containing peptides

Tyrosine phosphorylation and protein recognition, mediated by phosphotyrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PTK) p55(fyn) (Fyn). Peptide libraries were incubated for a short time in the presence of Fyn and phages displaying efficiently phosphorylated peptides were selected by panning over anti-phosphotyrosine antibodies. The characterization of the peptides enriched after three phosphorylation/selection rounds allowed us to define a canonical substrate sequence for the kinase Fyn, E-(phi/T)YGx phi, where phi represents any hydrophobic residue. A peptide conforming to this sequence is a better substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fyn SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, practically all the tyrosine containing peptides are phosphorylated, irrespective of their context. These "fully modified" peptide libraries are a valuable tool to study the specificity of phosphotyrosine mediated protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein Shc. Comparison of the peptide sequences permits us to confirm the essential role of N at position -3, while P often found at position -2 in natural targets is not absolutely required. Furthermore, our approach permits us to reveal an "extended" consensus indicating that residues that do not seem to influence binding in natural peptides can make productive contacts, at least in linear peptides.     

Features of construction and use of digital X-ray apparatuses for lung examinations

Comparative analysis for different antibodies' binding sites at the proteins samples, which were isolated from control and morphologically abnormal coniferous plants tissues have been investigated. Monoclonal antibodies against cytoskeletal proteins (actin, alpha- and beta tubulin), hsp 70 and against phosphotyrosine were used. It was detected that investigated biochemical features reflect morphological similarities between different plant species. Some differences between cytoskeleton proteins content in control and abnormal tissues have been also indicated. It is assumed that abnormal morphogenesis may be determined by low-level chronic irradiation during plants growth and development.     

Erythropoietin induces the association of phosphatidylinositol 3'-kinase with a tyrosine-phosphorylated protein complex containing the erythropoietin receptor

Stimulation of sensitive cells with erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the erythropoietin receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the erythropoietin-sensitive human UT7 cells by erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-erythropoietin bound to its receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated erythropoietin receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the erythropoietin receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated erythropoietin receptor.     

Enhanced expression of Ki-67, topoisomerase IIalpha, PCNA, p53 and p21WAF1/Cip1 reflecting proliferation and repair activity in UV-irradiated melanocytic nevi

To investigate the effect of ultraviolet (UV) irradiation on the expression of cell cycle-associated proteins, melanocytic nevi from healthy volunteers were partially covered, irradiated with a defined UV dose, and excised 1 week thereafter. The irradiated and the protected parts were examined separately by conventional microscopy and immunohistochemistry using the antibodies Ki-S11 (Ki-67), Ki-S7 (topoisomerase IIalpha), PC10 (proliferating cell nuclear antigen [PCNA]), DO-7 (p53), 6B6 (p21WAF1/Cip1), and the melanocytic marker HMB-45. DNA nick-end labeling was used as a marker of apoptosis. Irradiation resulted in morphological changes and increased HMB-45 reactivity. Proliferation, as assessed by Ki-67 and topoisomerase IIalpha expression, was also clearly enhanced in the UV-exposed areas. This was confirmed by the appearance of occasional mitotic figures. PCNA expression levels markedly exceeded those of the proliferation markers and did not correlate with the latter in most cases. p21 immunolabeling indices were also consistently augmented after UV exposure; hence it is likely that growth-inhibitory mechanisms partly compensate for the proliferative impulse, and the disproportional rise in PCNA expression probably reflects DNA repair activity. Enhanced p53 immunostaining in four cases suggests that the induction of p21 after irradiation may be p53 mediated, whereas no concomitant apoptotic events were observed. We conclude that UV light can stimulate the proliferative activity of melanocytes in melanocytic nevi, but that simultaneously cell cycle inhibitors are activated to permit DNA repair.     

Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock

Circadian clocks are synchronized by environmental cues such as light. Photoreceptor-deficient Arabidopsis thaliana mutants were used to measure the effect of light fluence rate on circadian period in plants. Phytochrome B is the primary high-intensity red light photoreceptor for circadian control, and phytochrome A acts under low-intensity red light. Cryptochrome 1 and phytochrome A both act to transmit low-fluence blue light to the clock. Cryptochrome 1 mediates high-intensity blue light signals for period length control. The presence of cryptochromes in both plants and animals suggests that circadian input pathways have been conserved throughout evolution.     

In vivo characterization of chimeric phytochromes in yeast

Phytochromes are plant photoreceptors that play a major role in photomorphogenesis. Two members of the phytochrome family have been characterized in some detail. Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings. Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs. We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*). SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA. Rice PHYA* did not undergo dark reversion in yeast. Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA. Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA*. These results are discussed with respect to structure/function analysis of phytochromes A and B.     

Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope

Self proteins are handled in the same way as foreign proteins by antigen presenting cells, but because of T-cell tolerance the presentation of self peptides does not normally lead to T cell activation. By providing physically linked T-cell help it is possible to overcome the B cell non-responsiveness toward self antigens. We have shown previously that a very potent antibody response, cross-reactive with a self protein, can be rapidly induced by immunizing with a recombinant immunogen consisting of the self protein with a foreign immunodominant T helper epitope inserted into its sequence (Dalum, I., Jensen, M. R., Hindersson, P., Elsner, H. I. and Mouritsen, S. (1996) J. Immnunol. 157, 4796). In this study we compare this approach for inducing autoantibodies against a self protein with the traditional method of conjugating the self antigen to a foreign carrier protein. The highly conserved self protein ubiquitin with an inserted epitope from ovalbumin (UbiOVA) is used as a model protein and compared to two traditionally conjugated immunogens consisting of ubiquitin chemically conjugated to a peptidic T helper epitope or to ovalbumin. The traditionally conjugated immunogens induce much slower and low titered ubiquitin specific antibody responses than the recombinant construct which also is capable of inducing antibodies directed against a much broader range of potential ubiquitin B cell determinants than the chemically conjugated immunogens. All three constructs are processed by antigen presenting cells and ovalbumin derived T cell epitopes are presented to T helper cells. From these observations it seems likely that the presence of non-shielded autologous B cell determinants on the immunogen is critical for the ability to induce a strong autoantibody response with a diverse fine specificity. Furthermore, the ubiquitin specific antibodies induced by UbiOVA contain higher levels of IgG2a/b relative to IgG1 compared to the conjugates. We therefore speculate that the insertion of a T cell epitope directly into the self antigen could possibly induce an immune response with a different Th1/Th2 balance than a response induced with traditional conjugates.     

Relationship of social support to stress responses and immune function in healthy and asthmatic adolescents

In animal species, spermiogenesis, the late stage of spermatogenesis, is characterized by a dramatic remodelling of chromatin which involves morphological changes and various modifications in the nature of the nuclear basic proteins. According to the evolution of species, three situations can be observed: a) persistence of somatic histones or appearance of sperm-specific histones; b) direct replacement of histones by generally smaller and more basic proteins called protamines; and c) occurrence of a double nuclear basic protein transition: histones are not directly replaced by protamines but by intermediate basic proteins which are themselves replaced by one or several protamines. However, in some species, two kinds of intermediate basic proteins can be distinguished in spermatid nuclei: transition proteins and protamine precursors. Whereas transition proteins are not structurally related either to histones or to protamines, protamine precursors are further processed at the end of spermiogenesis to give rise to the mature protamine. The molecular characteristics of the protamines as well as number of protamine types present in the spermatozoon vary from species to species. In some cases, protamine-encoding genes, although present, are not expressed to a significant level. The diversity and the precise function of intermediate basic proteins remain open to discussion. Some of them are the precursors of protamines but the mechanism, sequential or not, as well as the enzyme(s) involved in the proteolytic processing, remain to be discovered.