BBA - General Subjects (v.1760, #6)
Editorial Board (ii).
Effects of heating at neutral and acid pH on the structure of β-lactoglobulin A revealed by differential scanning calorimetry and circular dichroism spectroscopy by Ritsuko Wada; Yuki Fujita; Naofumi Kitabatake (841-847).
The structural change of β-lactoglobulin A (βLG A) on heating was measured at pH 3.0 and 7.5 with UV absorption difference spectra, differential scanning calorimetry (DSC), and circular dichroism (CD). At pH 3.0, βLG A showed a reversible structural change by heating at 80 °C, while an irreversible change was observed and molecular aggregates of βLG were formed by heating at 95 °C. DSC analysis of βLG A gave endothermic peaks at 75 °C and 90 °C at pH 7.5, and 90 °C at pH 3.0. At pH 7.5, βLG A modified with N-ethylmaleimide (NEM-βLG A) gave two endothermic peaks: at 72 °C and 90 °C. CD spectra of βLG A heated at various temperatures and pHs were measured and the spectra at pH 3.0 and 7.5 were not changed by heating to 95 °C and 80 °C, respectively. Unheated NEM-βLG A gave a spectrum similar to that of heated βLG A, suggesting that the secondary structure was changed by NEM treatment.
Keywords: β-lactoglobulin A (βLG A); N-ethylmaleimide (NEM); β-lactoglobulin A modified with NEM (NEM-βLG A); Differential scanning calorimetry (DSC); Circular dichroism (CD);
Developmental expression of two forms of arginase in Neurospora crassa by Gloria Ellen Turner; Richard L. Weiss (848-857).
N. crassa has two forms of arginase. The physiological role of multiple arginases is not understood. The two forms were shown to be differentially expressed from a single locus (aga) and both proteins are localized to the cytoplasm. The 36-kDa protein was expressed in minimal and arginine supplemented medium, whereas the 41-kDa form was detected only in the presence of arginine. In this study we examined developmental expression of the two arginase transcripts and proteins in conidia and during conidial germination. Two novel observations are revealed, storage of both arginase proteins in conidia and temporal expression of aga transcripts during early germination. To better understand the role of arginase in conidia and the nature of the temporal expression, we examined the effects of related metabolites, arginine, ornithine, proline, glutamate and glutamine on protein storage and temporal expression. These metabolites were used as supplements or sole nitrogen sources. Storage of arginase protein was detected in all conidial samples examined except when glutamate was used as the nitrogen source. The aga temporal RNA expression early in germination was abolished when arginine related metabolites were used as nitrogen sources. The exception to this result is observed with glutamate where temporal expression was seen when glutamate was the sole nitrogen source and abolished with glutamate supplementation. The temporal expression result supports a unique role for arginase in glutamate accumulation early in germination whereas the protein storage result supports the existence of a novel pathway utilizing arginase for glutamate synthesis in asexual spore development.
Keywords: Arginase; Neurospora crassa; Glutamate; Pyrroline-5-carboxylate dehydrogenase; Conidia;
Interaction of iron–sulfur flavoprotein with oxygen and hydrogen peroxide by Francisco Cruz; James G. Ferry (858-864).
The dimeric iron–sulfur flavoprotein (Isf) from Methanosarcina thermophila contains one 4Fe–4S center and one FMN per monomer, and is the prototype of a family widely distributed among strictly anaerobic prokaryotes. Although Isf is able to oxidize ferredoxin, the physiological electron acceptor is unknown; thus, the ability of Isf to reduce O2 and H2O2 was investigated. The product of O2 or H2O2 reduction by Isf was determined to be water. The kinetic parameters of the oxidative half-reactions with O2 and H2O2 as electron acceptors were consistent with a role for Isf in combating oxidative stress. Isf depleted of the 4Fe–4S cluster was unable to oxidize ferredoxin and reduce the FMN cofactor, supporting a role for the cluster in transfer of electrons from ferredoxin to the cofactor. The implications of these properties on the possible function and mechanism of Isf are discussed.
Keywords: Iron–sulfur flavoprotein; Oxygen; Hydrogen peroxide; Archaea; Methanosarcina thermophila; Oxidative stress;
Effects of molecular size and chemical structure on renal and hepatic removal of exogenously administered chondroitin sulfate in rats by Inah M.D. Pecly; Nelson M. Melo-Filho; Paulo A.S. Mourão (865-876).
Chondroitin sulfate, a glycosaminoglycan that is widely distributed among mammals, is used as a therapeutic agent in various diseases. Here, we focus on its absorption, excretion and tissue accumulation in rats. The concentration of 35S-chondroitin sulfate (35S-CS) in plasma reaches a peak in the first 5 min after intravenous administration and simultaneously increases in the urine. Approximately 25% of the 35S found in the urine appears as inorganic sulfate, indicating that 35S-CS is partially degraded during its renal filtration. The glycosaminoglycan is retained mainly by the liver and the kidney, where the amount of 35S reaches a plateau in the first 30 min, remains constant up to 2 h and then decreases markedly. Renal filtration and organ accumulation of 35S-CS decreases as the size of the glycosaminoglycan is reduced, especially in the liver. A derivative of 35S-CS that resists hyaluronidase digestion due to reduction of its glucuronic acid carboxyl groups appears at lower concentrations in plasma and in urine when compared with native 35S-CS. This derivative reaches higher levels in the kidney but lower levels in the liver when compared with the native molecule. Overall, our results indicate a balance between renal and hepatic mechanisms for removing chondroitin sulfate from plasma. The renal filtration increases as the molecular weight of the glycosaminoglycan decreases, whereas hepatic removal requires structural integrity and the presence of high-molecular-weight chains.
Keywords: Sulfated polysaccharide; Glycosaminoglycan; Renal filtration; Tissue accumulation; Plasma level;
Functional role of sepiapterin reductase in the biosynthesis of tetrahydropteridines in Dictyostelium discoideum Ax2 by Yong Kee Choi; Jin Sun Kong; Young Shik Park (877-882).
In Dictyostelium discoideum Ax2 l-erythro-tetrahydrobiopterin (BH4) is produced in much smaller amount than its stereoisomer d-threo-tetrahydrobiopterin (DH4), both of which are catalyzed by sepiapterin reductase (SR) at the terminal steps. In order to investigate their putative function and biosynthetic regulation, we performed quantitative analysis of not only the intracellular pteridines by HPLC but also the biosynthetic enzymes (GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase, SR, and aldose reductase-like enzyme) by Northern blot analysis and activity assay. We found that both SR transcript and activity increased in parallel with a remarkable decline in aldose reductase-like enzyme activity when BH4 increased transiently in the early development. Through in vitro assay of BH4/DH4 synthesis and in vivo rescue experiment of SR knockout mutant, we demonstrated that Dictyostelium SR favors DH4 synthesis while human SR does BH4 synthesis. The results suggest that Dictyostelium SR prefers 1′-oxo-2′-d-hydroxypropyl-tetrahydropterin to 6-pyruvoyltetrahydropterin as a substrate, thereby maintaining dominant production of DH4 over BH4 in sufficient supply of AR-like enzyme, while allowing increase of BH4 when SR prevails quantitatively over aldose reductase-like enzyme. On the other hand, a transient increase of BH4 may imply that BH4 has an independent function from DH4 in Dictyostelium.
Keywords: Dictyostelium; Tetrahydrobiopterin; Dictyopterin; Sepiapterin reductase; Developmental expression; Catalytic function;
A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins by Yoshiko Miura; Akio Sakaki; Masamichi Kamihira; Shinji Iijima; Kazukiyo Kobayashi (883-889).
A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs.
Keywords: Carbohydrate mimic peptide; Gb3; Phage display library; Shiga toxin;
Expression of hyaluronan synthase genes in umbilical cord blood stem/progenitor cells by Branka Grskovic; Christine Pollaschek; Mathias M. Mueller; Karl M. Stuhlmeier (890-895).
Scientific progress reveals an ever-expanding role of hyaluronan (HA) in diverse biological functions. It has become increasingly clear that HA might also be essential for certain functions of stem cells. CD133+ cells isolated from umbilical cord blood (UCB) seem to represent an alternative to CD34+ cells as a source of transplantable haematopoietic progenitor cells. The aim of this study was to investigate expression patterns of hyaluronan synthases (HAS) genes in freshly isolated and cultured UCB progenitor cells and to compare HAS mRNA levels to those found in non-progenitor cells. CD133+ stem cells were isolated from UCB using an immunomagnetic procedure. Investigation of HAS mRNA expression patterns in CD133+ and CD133− cells by RT-PCR was performed immediately after isolation as well as after cultivation towards myelomonocytic lineage. In addition, activation patterns of mitogen activated protein kinases (MAPK) were analyzed by Western blot experiments. mRNA for HAS1 is undetectable but HAS3 mRNA can be readily detected in freshly isolated CD133+ as well as in CD133− UCB cells. More importantly, our data demonstrate that mRNA for HAS2 can only be detected in CD133+ progenitor cells. In addition, while MAPK are slightly activated in CD133− UCB cells, no significant phosphorylation of MAPK could be observed in CD133+ cells, excluding a role of these kinases in the regulation of HAS2. HAS2 is expressed only in freshly isolated CD133+ cells and quickly diminishes during differentiation. Because of this, HAS2 gene expression might be suitable as a new marker for CD133+ UCB-derived stem cells.
Keywords: Umbilical cord blood (UCB); CD133+ cells; CD133− cells, hyaluronan synthase genes (HAS); Mitogen activated protein kinases (MAPK);
Involvement of the AtoS-AtoC signal transduction system in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli by Marina C. Theodorou; Christos A. Panagiotidis; Cynthia H. Panagiotidis; Anastasia A. Pantazaki; Dimitrios A. Kyriakidis (896-906).
The AtoS–AtoC signal transduction system in E. coli, which induces the atoDAEB operon for the growth of E. coli in short-chain fatty acids, can positively modulate the levels of poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles in E. coli. Increased amounts of cPHB were synthesized in E. coli upon exposure of the cells to acetoacetate, the inducer of the AtoS–AtoC two-component system. While E. coli that overproduce both components of the signal transduction system synthesize higher quantities of cPHB (1.5–4.5 fold), those that overproduce either AtoS or AtoC alone do not display such a phenotype. Lack of enhanced cPHB production was also observed in cells overexpressing AtoS and phosphorylation-impaired AtoC mutants. The results were not affected by the nature of the carbon source used, i.e., glucose, acetate or acetoacetate. An E. coli strain with a deletion in the atoS–atoC locus (ΔatoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the ΔatoSC strain was transformed with a plasmid carrying a 6.4-kb fragment encoding the AtoS–AtoC system, cPHB biosynthesis was restored to the level of the atoSC + cells. Introduction of a multicopy plasmid carrying a functional atoDAEB operon, but not one with a promoterless operon, resulted in increased cPHB synthesis only in atoSC + cells in the presence of acetoacetate. These results indicate that the presence of both a functional AtoS–AtoC two-component signal transduction system and a functional atoDAEB operon is critical for the enhanced cPHB biosynthesis in E. coli.
Keywords: Antizyme; atoDAEB; AtoS; AtoC; Two-component system; Poly-(R)-3-hydroxybutyrate;
Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein by Jennifer Bunce; David Achila; Evan Hetrick; Leighann Lesley; David L. Huffman (907-912).
Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an ∼72 amino acid consensus motif and the first four of these domains, denoted WLN1–4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5–6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5–6, and 6, denoted by WLN1, WLN3, WLN4, WLN5–6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.
Keywords: Wilson protein; ATP7B; Copper transfer; Metal-binding domain;
An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi by Leon A. Bouvier; Mariana R. Miranda; Gaspar E. Canepa; Maria Júlia M. Alves; Claudio A. Pereira (913-921).
Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.
Keywords: Trypanosoma cruzi; Adenylate kinase; Phosphotransferase; Arginine kinase; Chagas' disease;
Differences in the biophysical properties of membrane and cytoplasm of apoptotic cells revealed using dielectrophoresis by Fatima H. Labeed; Helen M. Coley; Michael P. Hughes (922-929).
We have used dielectrophoresis to determine the dielectric properties of human chronic myelogeneous leukaemic (K562) cells during apoptosis (programmed cell death). Our results indicate that K562 cells increase markedly in cytoplasmic conductivity from 0.28 S/m to 0.50 S/m within the first 4 h following treatment with staurosporine, which then lasts beyond 12 h, whilst cell shrinkage increases the capacitance of the membrane from 9.7 mF/m2 to 20 mF/m2. After 24 and 48 h of incubation with staurosporine, multiple sub-populations were detected, highlighted by the dielectric changes that the cell undergoes before death. By comparing these results with those obtained by common apoptosis monitoring techniques Annexin V and TMRE (tetramethylrhodamine ethylester), it is possible to infer the role of ion efflux in the progress of apoptosis. The use of dielectrophoresis for monitoring apoptosis offers a number of benefits as it is both rapid and non-invasive. It can also be used in parallel with other assays in high-throughput screening applications.
Keywords: Dielectrophoresis; Apoptosis; Cytoplasm; Annexin; TMRE; k562;
Glutamine synthetase degradation is controlled by oxidative proteolysis in the marine cyanobacterium Prochlorococcus marinus strain PCC 9511 by Guadalupe Gómez-Baena; Jose Manuel García-Fernández; Antonio López-Lozano; Fermín Toribio; Jesús Diez (930-940).
Prochlorococcus is one of the most important primary producers on Earth; its unusual features and ecological importance have made it a model organism, but nutrient assimilation has received little attention. Glutamine synthetase (GS) plays a key role in nitrogen metabolism and its central position justifies the fine regulation of this enzyme. The aim of this work is to demonstrate the involvement of metal-catalyzed oxidation (MCO) in the control of the biological activity and turnover of GS from Prochlorococcus. In order to study the physiological role of MCO, we have first characterized the in vitro biosynthetic inactivation and degradation of GS in the axenic PCC 9511 strain, testing then the effect of several stress conditions, such as the presence of electron transport inhibitors, darkness and aging, on the inactivation and degradation of GS. It is noteworthy that the physiological substrates of GS could protect the enzyme from the oxidative inactivation and ATP partially reverted this inactivation once the enzyme had been oxidized, being this effect higher in the presence of glutamate. We have also found that the GS from aged cultures is degraded to the same smaller size fragments obtained in the in vitro degradation of GS by an oxidative model system (Fe3+/NADH/NADH oxidase/O2). These results suggest the implication of MCO in the age- and oxidative state-dependent degradation of GS from Prochlorococcus.
Keywords: Glutamine synthetase; Metal-catalyzed oxidation; Proteolysis; Physiological regulation; Marine cyanobacteria;
Extracellular signal regulated MAP-kinase signalling in osmotically stressed trout hepatocytes by Hannes L. Ebner; Birgit Fiechtner; Bernd Pelster; Gerhard Krumschnabel (941-950).
Activation of the extracellular signal-regulated MAP-kinase (ERK) by anisoosmotic conditions, the underlying signalling pathways, and the role of protein kinases in cell volume regulation were investigated in trout hepatocytes. While hyperosmolarity left phosphorylated ERK (pERK) levels unaffected, hypoosmolarity caused a significant increase of pERK within 2 min which peaked at around 30 min. Chelating extracellular Ca2+ to prevent the influx of Ca2+ associated with swelling reduced iso- and abolished hypoosmotic ERK activation. Similarly, inhibiting the ERK activator MEK, tyrosine kinases, or PKC inhibited the increase of pERK. In contrast, exposing cells to chelerytrine or staurosporine, PKC inhibitors of little specificity, increased pERK independently from osmotic conditions. Blocking PI3 kinase, application of 8-Br-cAMP, exposure to a P-receptor antagonist, and inhibition of p38 MAP-kinase had no effect on ERK activity. A significant reduction of regulatory volume decrease (RVD) after hypoosmotic swelling caused by MEK-inhibition and an even more pronounced reduction due to p38 inhibition indicates a role for MAP-kinases in volume regulation, but a lack of correlation between the impact of protein kinase inhibitors on pERK levels and on RVD suggests that ERK may merely modulate volume recovery. Immunocytochemical detection of pERK indicated cytoplasmic activation, but no nuclear accumulation within 30 min, supporting the notion that ERK exerts non-genomic effects. Overall, our data underscore the complexity of hypoosmotic ERK signalling and suggest a role of ERK and p38 in acute cell volume regulation.
Keywords: Trout hepatocyte; MAP kinase; ERK; Volume regulation; Calcium; PKC;
Recognition characters in peptide–polyphenol complex formation by T. Richard; D. Lefeuvre; A. Descendit; S. Quideau; J.P. Monti (951-958).
Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein–polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.
Keywords: Bradykinin; Polyphenol; NMR;
Epitope mapping of the domains of human angiotensin converting enzyme by Elena V. Kugaevskaya; Ekaterina F. Kolesanova; Sergey A. Kozin; Alexander V. Veselovsky; Ilya R. Dedinsky; Yulia E. Elisseeva (959-965).
Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning (“PEPSCAN”) approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.
Keywords: ACE; Domains; Antigen determinants; Structure;
FBXO25, an F-box protein homologue of atrogin-1, is not induced in atrophying muscle by Ana Leticia G.C. Maragno; Munira M.A. Baqui; Marcelo D. Gomes (966-972).
Atrogin-1/MAFbx/FBXO32 is a muscle-specific ubiquitin-ligase (E3) that is dramatically increased in atrophying muscle. Here, we have investigated the functional relationship between atrogin-1 and FBXO25 which shares 65% amino acid identity. Using a RT-PCR, we demonstrated that FBXO25 is highly expressed in brain, kidney, and intestine, whereas atrogin-1 expression is largely restricted to striate muscle. FBXO25 was shown here to contain a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the major components of SCF-type E3s. In addition, the productive SCF complex containing FBXO25 showed ubiquitin ligase activity. We investigated the differential expression of atrogin-1 and FBXO25 in fasted and dexamethasone-treated mice and also in rats with streptozotocin-induced diabetes. Although the atrogin-1 was strongly induced in muscle in all three models, no changes were observed in the expression of FBXO25. Therefore, here we have shown that FBXO25 is a novel F-box protein analogous to atrogin-1, which is not involved in muscle atrophy. Further functional studies should elucidate the exact role of FBXO25 in the ubiquitin-proteasome pathway.
Keywords: F-box; FBXO25; Atrogin-1; Atrophy; Ubiquitin; Proteasome;
Modulation of polyene antibiotics self-association by ions from the Hofmeister series by Mercedes Toledo Grijalba; Monique Chéron; Edward Borowski; Jacques Bolard; Shirley Schreier (973-979).
The toxicity of the antifungal polyene antibiotic amphotericin B (AMB) has been related to its low solubility, more specifically to a self-associated form termed toxic aggregate. In addition, AMB in aqueous medium gives rise to concentration, ionic strength, and time-dependent polydisperse systems. For this reason different approaches, including the use of several lipid aggregates, have been used in attempts to improve the drug's solubility and increase its therapeutic index. In this context, understanding AMB's self-association properties should help in the preparation of less toxic formulations. Ions from the Hofmeister series alter water properties: while kosmotropes (water structure makers—sulfate, citrate, phosphate) decrease solute solubility, chaotropes (water structure breakers—perchlorate, thiocyanate, trichloroacetate, and the neutral molecule urea) have opposite effects. This work reports a study of the effect of Hofmeister ions and urea on the self-aggregation of AMB and some of its derivatives. Optical absorption and circular dichroism spectra were used to monitor monomeric and aggregated antibiotic. While kosmotropes increased aggregation in a concentration-dependent manner, the opposite was observed for chaotropes. It is shown, for the first time, that thiocyanate and trichloroacetate can induce complete AMB monomerization. The understanding of these processes at the physicochemical and molecular levels and the possibility of modulating the aggregation state of AMB and its derivatives should contribute to elucidate the mechanisms of action and toxicity of this widely used antibiotic and to develop more efficient and less toxic preparations.
Keywords: Amphotericin B; Polyene antibiotic; Self-association; Solubility; Toxicity; Hofmeister ions; Water structure;
Normal distribution of presenilin-1 and nicastrin in skeletal muscle and the differential responses of these proteins after denervation by Kunihiro Sakuma; Ryuta Nakao; Yuka Yamasa; Masahiro Yasuhara (980-987).
Presenilin-1 and nicastrin, two components of γ-secretase associated with Alzheimer's disease plaques, are present in the synapses of the brain and in various peripheral organs, including skeletal muscle. In the present study, we examined the expression pattern of presenilin-1 and nicastrin in normal and denervated hindlimb muscles of the rat. Using immunohistochemical approaches, we found that presenilin-1 and AChRα was co-localized at the neuromuscular junction in the normal skeletal muscles of rats. The immunoreactivities of both presenilin-1 and nicastrin were also observed at the sarcolemma of muscle fibers. We discovered that presenilin-1 mRNA and its protein are upregulated after denervation of the soleus and tibialis anterior muscles. Furthermore, clear co-localization between presenilin-1 and DAPI, but not nicastrin, was noted in several myonuclei in the denervated muscles. We recognized a few fibers possessing both ubiquitin and presenilin-1 protein in the cytosol. The amount of presenilin-1 in the nucleus and membrane fraction was more abundantly expressed in the denervated muscle fibers. In contrast, no significant difference in the nicastrin protein level was observed between normal and denervated muscle fibers. These data suggest that enhanced presenilin-1 protein may play a role in the degeneration and regeneration of skeletal muscle.
Keywords: Presenilin-1; Nicastrin; Skeletal muscle; Denervation; Myonucleus;