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1.
Genet Med ; 26(3): 101051, 2024 03.
Article in English | MEDLINE | ID: mdl-38131308

ABSTRACT

PURPOSE: The UK 100,000 Genomes Project offered participants screening for additional findings (AFs) in genes associated with familial hypercholesterolemia (FH) or hereditary cancer syndromes including breast/ovarian cancer (HBOC), Lynch, familial adenomatous polyposis, MYH-associated polyposis, multiple endocrine neoplasia (MEN), and von Hippel-Lindau. Here, we report disclosure processes, manifestation of AF-related disease, outcomes, and costs. METHODS: An observational study in an area representing one-fifth of England. RESULTS: Data were collected from 89 adult AF recipients. At disclosure, among 57 recipients of a cancer-predisposition-associated AF and 32 recipients of an FH-associated AF, 35% and 88%, respectively, had personal and/or family history evidence of AF-related disease. During post-disclosure investigations, 4 cancer-AF recipients had evidence of disease, including 1 medullary thyroid cancer. Six women with an HBOC AF, 3 women with a Lynch syndrome AF, and 2 individuals with a MEN AF elected for risk-reducing surgery. New hyperlipidemia diagnoses were made in 6 FH-AF recipients and treatment (re-)initiated for 7 with prior hyperlipidemia. Generating and disclosing AFs in this region cost £1.4m; £8680 per clinically significant AF. CONCLUSION: Generation and disclosure of AFs identifies individuals with and without personal or familial evidence of disease and prompts appropriate clinical interventions. Results can inform policy toward secondary findings.


Subject(s)
Breast Neoplasms , Hyperlipidemias , Neoplastic Syndromes, Hereditary , Adult , Humans , Female , Genetic Testing/methods , Disclosure , Neoplastic Syndromes, Hereditary/genetics , Breast Neoplasms/genetics , Hyperlipidemias/genetics , Delivery of Health Care , Genetic Predisposition to Disease
2.
Br J Clin Pharmacol ; 87(6): 2542-2548, 2021 06.
Article in English | MEDLINE | ID: mdl-33217039

ABSTRACT

AIMS: Asparaginase (ASP) hypersensitivity is a well-known challenge in the treatment of lymphoblastic malignancies. In terms of cost considerations, the cheap native Escherichia coli ASP, the most immunogenic form of this medication, is used in the first line in middle-income countries. Previously, the role of the HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02 haplotype had been established to associate with E. coli ASP hypersensitivity. We investigated a possible cost-effective genetic testing method to identify patients harbouring the risk HLA haplotype in order to pave the way for safer ASP treatment. METHODS: In 241 patients with previously determined HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02 haplotype and known ASP hypersensitivity status, 4 candidate HLA-tagging single-nucleotide polymorphisms (SNP)s were measured, and the performance of the different sets of these tag SNPs was evaluated. RESULTS: We identified a combination of 2 SNPs - rs28383172 and rs7775228 - as a tag for HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02 haplotype with sensitivity and specificity values >95%. In line with previous findings, we found complete concordance between HLA-DRB1*07:01 and rs28383172. With bioinformatics methods, the results were also confirmed in the 1000 Genomes dataset in different ethnic groups. CONCLUSION: Rs28383172 and rs7775228 are suitable for identifying HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02 carriers. Compared to the rest of the population, patients with hypersensitivity-prone genotype would benefit more from the administration of less immunogenic PEGylated ASP before the hypersensitivity evolves, incurring minimal extra cost.


Subject(s)
Asparaginase , Drug Hypersensitivity , HLA-DRB1 Chains , Humans , Alleles , Asparaginase/adverse effects , Drug Hypersensitivity/genetics , Escherichia coli , Gene Frequency , Genetic Predisposition to Disease , Haplotypes , HLA-DQ alpha-Chains/genetics , HLA-DQ beta-Chains/genetics , HLA-DRB1 Chains/genetics , Polymorphism, Single Nucleotide
3.
J Mol Med (Berl) ; 98(7): 963-971, 2020 07.
Article in English | MEDLINE | ID: mdl-32435918

ABSTRACT

Preventing the ototoxicity caused by cisplatin is a major issue yet to be overcome. Useful preventive treatments will soon be available. Consequently, the next step is to filter out those patients who are more prone to develop ototoxicity. The aim of this study was to prospectively evaluate potential predictive markers of acute ototoxicity as determined by measures of distortion product otoacoustic emissions (DPOAEs). A total of 118 patients from our previous DPOAE analysis were put under evaluation. Ototoxic cases were divided according to unilateral (n = 45) or bilateral (n = 23) involvement. The clinicopathological characteristics, hearing test results, germline GSTT1, GSTM1, and GSTP1 polymorphisms, and common laboratory parameters were included in the new analysis. Univariate and multivariate statistical tests were applied. According to multivariate logistic regression, the only independent predictor of unilateral ototoxicity (vs. non-affected) was a GSTM1 null genotype (OR = 4.52; 95%CI = 1.3-16.3), while for bilateral damage, the GSTT1 null genotype (OR = 4.76; 1.4-16) was a predictor. The higher starting serum urea level was characteristic of bilateral ototoxicity; however, the only independent marker of bilateral (vs. unilateral) ototoxicity was the presence of GSTT1 null genotype (OR = 2.44; 1.23-4.85). Different processes, involving the GSTM1 and GSTT1 genotypes, respectively, govern the development of acute unilateral and bilateral ototoxicities. Further research is needed to clarify these processes. Based on the above findings, patients whom are at risk may be selected for otoprotective therapies. KEY MESSAGES: The acute ototoxicity was determined by DPOAE in 118 testicular cancer patients. GSTM1 null was the only marker of unilateral ototoxicity (vs. non-affected). The only marker of bilateral hearing loss (vs. non-affected) was the GSTT1 null. GSTT1 null was also the marker of bilateral vs. unilateral ototoxicity. A high-risk group may be selected for new, individualized otoprotective treatment.


Subject(s)
Cisplatin/adverse effects , Glutathione Transferase/genetics , Ototoxicity/etiology , Ototoxicity/genetics , Adult , Biomarkers/metabolism , Case-Control Studies , Gene Frequency/genetics , Genotype , Germ Cells/metabolism , Hearing Tests/methods , Humans , Male , Neoplasms, Germ Cell and Embryonal/drug therapy , Neoplasms, Germ Cell and Embryonal/genetics , Ototoxicity/metabolism , Polymorphism, Genetic/genetics , Prospective Studies , Testicular Neoplasms/drug therapy , Testicular Neoplasms/genetics
4.
Orv Hetil ; 159(32): 1295-1302, 2018 Aug.
Article in Hungarian | MEDLINE | ID: mdl-30078355

ABSTRACT

Etoposide is a topoisomerase II inhibitor antitumor agent which is widely used in the treatment of several hematologic malignancies and solid tumors. The therapeutic index of etoposide is quite high, thus its application causes several short-term and long-term side effects which can decrease the chance to cure patients. Drug dosing is based on body surface area calculation; recommendations for individual dosing do not exist yet. The biotransformation and transportation of etoposide are carried out by enzymes and transporters as reported in pharmacogenomic studies published in this area. Nowadays pharmacoepigenetics research has come to the fore. The authors wish to give an insight into the research of the epigenetical changes of the etoposide pathways, especially focusing on published findings on enzymes and transporters with pharmacokinetic relevance. In the future, epigenetical changes of the etoposide pathway might have a great role in diagnostics, prognostics and personalized medicine. Orv Hetil. 2018; 159(32): 1295-1302.


Subject(s)
Antineoplastic Agents, Phytogenic/metabolism , Biological Transport/genetics , Epigenesis, Genetic , Etoposide/metabolism , Antineoplastic Agents, Phytogenic/pharmacokinetics , Etoposide/pharmacokinetics , Human Body , Humans
5.
Int J Syst Evol Microbiol ; 67(11): 4742-4746, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28950929

ABSTRACT

A new betaproteobacterium, CGI-09T, was isolated from an activated sludge bioreactor which treated landfill leachate. Based on 16S rRNA gene sequence analysis, the new strain shared the highest pairwise similarity values with members of the order Burkholderiales: Derxia gummosa IAM 13946T (family Alcaligenaceae), 93.7 % and Lautropia mirabilis DSM 11362T (family Burkholderiaceae), 93.6 %. Cells of strain CGI-09T were rod-shaped and non-motile. The new strain was oxidase and catalase positive and capable of reducing nitrate to nitrite. The predominant fatty acids were C16 : 1 ω7c, C16 : 0, cycloC17 : 0 and C18 : 1 ω7c, the major respiratory quinone was Q-8, and the detected polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and an unknown phospholipid. The G+C content of the genomic DNA of strain CGI-09T was 70.2 mol%. The new bacterium can be distinguished from the members of genera Derxia and Lautropia based on its non-motile cells, arginine dihydrolase activity, its high cyclo C17 : 0 fatty acid content and the lack of hydroxy fatty acids. On the basis of the phenotypic, chemotaxonomic and molecular data, strain CGI-09T is considered to represent a new genus and species within the family Burkholderiaceae, for which the name Quisquiliibacterium transsilvanicum gen. nov., sp. nov. is proposed. The type strain is CGI-09T (=DSM 29781T=JCM 31785T).


Subject(s)
Alcaligenaceae/classification , Bioreactors/microbiology , Phylogeny , Sewage/microbiology , Alcaligenaceae/genetics , Alcaligenaceae/isolation & purification , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Phospholipids/chemistry , RNA, Ribosomal, 16S/genetics , Romania , Sequence Analysis, DNA , Ubiquinone/chemistry
6.
Mol Cell Biol ; 35(17): 3083-102, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26124280

ABSTRACT

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.


Subject(s)
ErbB Receptors/genetics , ErbB Receptors/ultrastructure , Amino Acid Sequence , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Crystallography, X-Ray , Enzyme Activation/genetics , ErbB Receptors/metabolism , Flow Cytometry , HEK293 Cells , Humans , Molecular Dynamics Simulation , Phosphorylation , Protein Structure, Tertiary , Sequence Deletion/genetics
7.
Annu Rev Biochem ; 84: 739-64, 2015.
Article in English | MEDLINE | ID: mdl-25621509

ABSTRACT

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a critical role in the pathogenesis of many cancers. The structure of intact forms of this receptor has yet to be determined, but intense investigations of fragments of the receptor have provided a detailed view of its activation mechanism, which we review here. Ligand binding converts the receptor to a dimeric form, in which contacts are restricted to the receptor itself, allowing heterodimerization of the four EGFR family members without direct ligand involvement. Activation of the receptor depends on the formation of an asymmetric dimer of kinase domains, in which one kinase domain allosterically activates the other. Coupling between the extracellular and intracellular domains may involve a switch between alternative crossings of the transmembrane helices, which form dimeric structures. We also discuss how receptor regulation is compromised by oncogenic mutations and the structural basis for negative cooperativity in ligand binding.


Subject(s)
ErbB Receptors/metabolism , Animals , Dimerization , Epidermal Growth Factor/metabolism , ErbB Receptors/chemistry , Humans , Protein Binding , Protein Structure, Tertiary
8.
Orv Hetil ; 155(25): 986-92, 2014 Jun 22.
Article in Hungarian | MEDLINE | ID: mdl-24936574

ABSTRACT

INTRODUCTION: During chemotherapy of cancer patients, vein inflammation may develop which may lead to pain and difficulty in blood sampling. The use of implanted venous access ports may overcome these problems. With a correct use of needles, venous port membrane may be pierced 2000-2500 times. AIM: The aim of the authors was to analyze their experience with venous access ports and evaluate whether insertion of venous ports occurred in an optimal time. METHOD: A retrospective study of 63 patients who received venous access ports between 2005 and 2012 was carried out. Complication rate and the time of venous port insertion were analysed. RESULTS: Of the 63 patients, the venous access port was removed in 22 patients because it was no longer required (n = 14) due to the development of complications (n = 8). 24 patients died from the underlying disease and 17 patients are still alive with the venous port in place. CONCLUSIONS: In the period studied patients with venous access ports had an improved quality of life. However, the authors conclude that venous access ports were not inserted in an optimal time.


Subject(s)
Antineoplastic Agents/administration & dosage , Catheterization, Central Venous , Catheters, Indwelling , Neoplasms/drug therapy , Adult , Aged , Catheterization, Central Venous/adverse effects , Catheterization, Central Venous/statistics & numerical data , Catheters, Indwelling/adverse effects , Catheters, Indwelling/statistics & numerical data , Female , Humans , Male , Middle Aged , Quality of Life , Retrospective Studies , Time Factors
9.
Appl Biochem Biotechnol ; 170(4): 819-30, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23613116

ABSTRACT

The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.


Subject(s)
Protein Folding , Protein Multimerization , Tumor Necrosis Factors/metabolism , Brain/cytology , Brain/metabolism , DNA, Complementary/genetics , DNA, Complementary/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Glucocorticoid-Induced TNFR-Related Protein/metabolism , Humans , Inclusion Bodies/metabolism , Ligands , Phosphorylation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tumor Necrosis Factors/genetics , U937 Cells
10.
Cell ; 152(3): 543-56, 2013 Jan 31.
Article in English | MEDLINE | ID: mdl-23374349

ABSTRACT

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.


Subject(s)
Cell Membrane/metabolism , Epidermal Growth Factor/metabolism , ErbB Receptors/chemistry , Signal Transduction , Animals , COS Cells , Cell Membrane/chemistry , Chlorocebus aethiops , Dimerization , ErbB Receptors/metabolism , Humans , Models, Molecular
11.
Curr Opin Struct Biol ; 21(6): 777-84, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21868214

ABSTRACT

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in cell growth that is often misregulated in cancer. Several recent studies highlight the unique structural mechanisms involved in its regulation. Some elucidate the important role that the juxtamembrane segment and the transmembrane helix play in stabilizing the activating asymmetric kinase dimer, and suggest that its activation mechanism is likely to be conserved among the other human EGFR-related receptors. Other studies provide new explanations for two long observed, but poorly understood phenomena, the apparent heterogeneity in ligand binding and the formation of ligand-independent dimers. New insights into the allosteric mechanisms utilized by intracellular regulators of EGFR provide hope that allosteric sites could be used as targets for drug development.


Subject(s)
ErbB Receptors/chemistry , ErbB Receptors/metabolism , Allosteric Regulation , Allosteric Site , Catalysis , Cell Membrane/metabolism , Dimerization , Epidermal Growth Factor/metabolism , Humans , Ligands , Models, Molecular , Protein Conformation
12.
Biochem Biophys Res Commun ; 401(2): 281-6, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20851108

ABSTRACT

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca2+ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca2+ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca2+ release channel (RyR1). Here we report that SPC also displaces Ca2+-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca2+CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [3H]ryanodine binding assays. In the presence of Ca2+CaM, however, the addition of SPC did not reduce [3H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca2+CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca2+ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.


Subject(s)
Calmodulin/metabolism , Muscle, Skeletal/metabolism , Phosphorylcholine/analogs & derivatives , Ryanodine Receptor Calcium Release Channel/metabolism , Sphingosine/analogs & derivatives , Animals , Muscle, Skeletal/drug effects , Permeability , Phosphorylcholine/metabolism , Phosphorylcholine/pharmacology , Rabbits , Sphingosine/metabolism , Sphingosine/pharmacology
13.
FASEB J ; 24(10): 3829-39, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20522785

ABSTRACT

Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.


Subject(s)
Calmodulin/metabolism , Signal Transduction , Sphingolipids/metabolism , Animals , Brain/metabolism , Calorimetry , Cattle , Crystallography, X-Ray , Fluorescence , Kinetics , Micelles , Microsomes/metabolism , Models, Molecular , Protein Binding
14.
Proc Natl Acad Sci U S A ; 107(12): 5429-34, 2010 Mar 23.
Article in English | MEDLINE | ID: mdl-20212158

ABSTRACT

Numerous human genes display dual coding within alternatively spliced regions, which give rise to distinct protein products that include segments translated in more than one reading frame. To resolve the ensuing protein structural puzzle, we identified 67 human genes with alternative splice variants comprising a dual-coding region at least 75 nucleotides in length and analyzed the structural status of the protein segments they encode. The inspection of their amino acid composition and predictions by the IUPred and PONDR VSL2 algorithms suggest a high propensity for structural disorder in dual-coding regions. In the case of +1 frameshifts, the average level of disorder in the two frames is similarly high (47.2% in the ancestral frame, 58.2% in the derived frame, with the average level of disorder in human proteins being approximately 30%), whereas in the case of -1 frameshifts, there is a significant tendency to become more disordered upon shifting the frame (16.7% in the ancestral frame, 56.3% in the derived frame). The regions encoded by the derived frame are mostly disordered (disorder percentage > 50%) in 39 out of 62 cases, which strongly suggests that structural disorder enables these protein products to exist and function without the need of a highly evolved 3D fold. The potential advantages are also demonstrated by the appearance of novel functions and the high incidence of transcripts escaping nonsense-mediated decay. By discussing several examples, we demonstrate that dual coding may be an effective mechanism for the evolutionary appearance of novel intrinsically disordered regions with new functions.


Subject(s)
Alternative Splicing , Proteins/chemistry , Proteins/genetics , Reading Frames , Amino Acids/analysis , Biophysical Phenomena , Chromogranins , Codon, Nonsense , Computational Biology , Cyclin-Dependent Kinase Inhibitor p16/chemistry , Cyclin-Dependent Kinase Inhibitor p16/genetics , Databases, Genetic , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/genetics , Humans , Protein Biosynthesis , Protein Folding , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism
15.
J Biol Chem ; 285(3): 1799-808, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-19910470

ABSTRACT

Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427-437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca2+ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca2+ signaling, we hypothesize that SPC could lead to Ca2+ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca2+ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca2+-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors.


Subject(s)
Calcium Signaling/drug effects , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Phosphorylcholine/analogs & derivatives , Sphingosine/analogs & derivatives , Amino Acid Sequence , Animals , Apoproteins/metabolism , Binding, Competitive , Calcium/metabolism , Calmodulin/chemistry , Cattle , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Kinetics , Melitten/chemistry , Melitten/metabolism , Micelles , Molecular Sequence Data , Phosphorylcholine/metabolism , Phosphorylcholine/pharmacology , Protein Structure, Tertiary , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/metabolism , Spectrometry, Fluorescence , Sphingosine/metabolism , Sphingosine/pharmacology
16.
Orv Hetil ; 150(27): 1251-7, 2009 Jul 05.
Article in Hungarian | MEDLINE | ID: mdl-19531458

ABSTRACT

UNLABELLED: Five years ago (in September, 2003), the activity of the 5th Haemopoietic Stem Cell Transplantation Centre of Hungary has begun. This centre has been registered as No 648. by the European Group for Blood and Marrow Transplantation-Centres. AIMS: To supply the needs of stem cell transplantation regions in north-east Hungary and to develop an active co-operation with the Hungarian and international centres. METHODS: Transplantations were made according to international criteria. RESULTS: 150 autologous stem cell transplantations has been performed so far, including 74 patients with myeloma multiplex, 43 patients with non-Hodgkin lymphoma, 27 patients with Hodgkin's disease, 4 patients with autoimmune disease, and one patient with leiomyosarcoma. The survival rates were similar to the previous Hungarian and international data. The centre played a role in other activities using stem cell therapy at the University of Debrecen (dendritic cell vaccine program, stem cell therapy in myocardial infarction, stem cell therapy in peripheral arterial- and autoimmune diseases). This centre performed the largest quantity of the conditioning protocol Zevalin, Bischloronitrosourea, Etoposide, cytosine-Arabinoside, Melphalan in non-Hodgkin lymphoma in Hungary; ten patients were treated with this protocol.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Hematopoietic Stem Cell Transplantation , Transplantation Conditioning/methods , Antibodies, Monoclonal/administration & dosage , Autoimmune Diseases/surgery , Cytarabine/administration & dosage , Etoposide/administration & dosage , Hodgkin Disease/surgery , Humans , Hungary , International Cooperation , Kaplan-Meier Estimate , Leiomyosarcoma/surgery , Lymphoma, Non-Hodgkin/surgery , Melphalan/administration & dosage , Multiple Myeloma/surgery , Nitrosourea Compounds/administration & dosage , Time Factors , Transplantation, Autologous , Treatment Outcome
17.
Biochemistry ; 48(24): 5689-99, 2009 Jun 23.
Article in English | MEDLINE | ID: mdl-19432419

ABSTRACT

Beta(2)-microglobulin- (beta2m-) based fibril deposition is the key symptom in dialysis-related amyloidosis. beta2m readily forms amyloid fibrils in vitro at pH 2.5. However, it is not well understood which factors promote this process in vivo, because beta2m cannot polymerize at neutral pH without additives even at elevated concentration. Here we show that lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator, promotes amyloid formation under physiological conditions through a complex mechanism. In the presence of LPA, at and above its critical micelle concentration, native beta2m became sensitive to limited proteolytic digestion, indicating increased conformational flexibility. Isothermal titration calorimetry indicates that beta2m exhibits high affinity for LPA. Fluorescence and CD spectroscopy, as well as calorimetry, showed that LPA destabilizes the structure of monomeric beta2m inducing a partially unfolded form. This intermediate is capable of fibril extension in a nucleation-dependent manner. Our findings also indicate that the molecular organization of fibrils formed under physiological conditions differs from that of fibrils formed at pH 2.5. Fibrils grown in the presence of LPA depolymerize very slowly in the absence of LPA; moreover, LPA stabilizes the fibrils even below its critical micelle concentration. Neither the amyloidogenic nor the fibril-stabilizing effects of LPA were mimicked by its structural and functional lysophospholipid analogues, showing its selectivity. On the basis of our findings and the observed increase in blood LPA levels in dialysis patients, we suggest that the interaction of LPA with beta2m might contribute to the pathomechanism of dialysis-related amyloidosis.


Subject(s)
Amyloid/chemistry , Lysophospholipids/pharmacology , beta 2-Microglobulin/chemistry , Amyloid/metabolism , Amyloid/ultrastructure , Binding Sites , Circular Dichroism , Hydrogen-Ion Concentration , Models, Molecular , Osmolar Concentration , Protein Folding , Protein Structure, Secondary , Temperature , beta 2-Microglobulin/metabolism , beta 2-Microglobulin/ultrastructure
18.
J Plant Physiol ; 166(1): 72-9, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-18430489

ABSTRACT

Beneficial effects of low-concentration chemical stressors have been investigated previously in different model systems. The symptoms of stimulation are known from earlier studies, but information about the mechanism is at an initial stage. In the present work, the mechanism of stimulation of low-concentration Cd (5 x 10(-8)M) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 10(-7)M) was investigated in barley seedlings. In treated plants, the amount of cytokinins increased in roots and, after being transported to the leaves, they caused stimulation there. To identify the signal transduction pathway(s) involved in the primary stimulation of cytokinin synthesis (and/or activation) in roots, specific phosphatidylinositol-4,5-bisphosphate-inositol-1,4,5-triphosphate/diacylglycerol (PIP(2)-IP(3)/DAG) and mitogen activated protein kinase (MAPK) signaling pathway inhibitors were added to the nutrient solution, and all proved to be effective, eliminating the stimulation by the stressors. Measurements of superoxide dismutase (SOD, EC 1.15.1.1) activity and the amount of malonyldialdehyde (MDA) showed that the increased amount of Cd did not cause oxidative stress in the roots, and no oxidative stress was found in the leaves, where Cd did not even accumulate. DCMU slightly increased the activity of SOD after 1 week in roots, but did not cause lipid peroxidation. In leaves, there was no oxidative stress upon treatment with DCMU. Thus, oxidative stress cannot be responsible for the stimulation with low-concentration stressors, as they changed the activity of SOD differently, while being equally stimulative for the plants.


Subject(s)
Hordeum/metabolism , Seedlings/metabolism , Stress, Physiological , Cadmium/toxicity , Chlorophyll/metabolism , Chloroplasts/drug effects , Chloroplasts/enzymology , Chloroplasts/ultrastructure , Cotyledon/drug effects , Cotyledon/enzymology , Cytokinins/metabolism , Diuron/pharmacology , Hordeum/drug effects , Hordeum/enzymology , Isoenzymes/metabolism , Malondialdehyde/metabolism , Models, Biological , Plant Leaves/drug effects , Plant Leaves/enzymology , Plant Roots/drug effects , Plant Roots/enzymology , Seedlings/drug effects , Seedlings/enzymology , Stress, Physiological/drug effects , Superoxide Dismutase/metabolism
19.
Biochem J ; 410(2): 427-37, 2008 Mar 01.
Article in English | MEDLINE | ID: mdl-17979830

ABSTRACT

S1P (sphingosine 1-phosphate) and SPC (sphingosylphosphorylcholine) have been recently recognized as important mediators of cell signalling, regulating basic cellular processes such as growth,differentiation, apoptosis, motility and Ca2+ homoeostasis.Interestingly, they can also act as first and second messengers. Although their activation of cell-surface G-protein-coupled receptors has been studied extensively, not much is known about heir intracellular mechanism of action, and their target proteins are yet to be identified. We hypothesized that these sphingolipids might bind to CaM (calmodulin), the ubiquitous intracellular Ca2+sensor. Binding assays utilizing intrinsic tyrosine fluorescence of the protein, dansyl-labelled CaM and surface plasmon resonance revealed that SPC binds to both apo- and Ca2+-saturated CaM selectively, when compared with the related lysophospholipid mediators S1P, LPA (lysophosphatidic acid) and LPC (lysophosphatidylcholine). Experiments carried out with the model CaM-binding domain melittin showed that SPC dissociates the CaM-target peptide complex, suggesting an inhibitory role. The functional effect of the interaction was examined on two target enzymes, phosphodiesterase and calcineurin, and SPC inhibited the Ca2+/CaM-dependent activity of both. Thus we propose that CaM might be an intracellular receptor for SPC, and raise the possibility of a novel endogenous regulation of CaM.


Subject(s)
Calmodulin/antagonists & inhibitors , Phosphorylcholine/analogs & derivatives , Sphingosine/analogs & derivatives , Animals , Brain Chemistry , Cattle , Liposomes , Micelles , Phosphorylcholine/isolation & purification , Signal Transduction , Sphingosine/isolation & purification , Sphingosine/physiology , Tyrosine/analysis
20.
J Hazard Mater ; 105(1-3): 39-60, 2003 Dec 12.
Article in English | MEDLINE | ID: mdl-14623419

ABSTRACT

Facilities using hazardous substances are required to comply with risk management programs that aim to reduce the frequency of chemical accidents and the severity of consequences in the event of an accident. Both the European Union and the United States use chemical-specific weight thresholds for toxic substances to determine those facilities and processes that must comply with such programs. This study evaluates whether the establishment and use of these 'threshold quantities' is consistent and protective of public health. The chemical footprint or hazard zone length is calculated using current threshold quantities and 'level of concern' (LOC) concentrations for 77 toxic chemicals in the US regulations. Using the worst-case scenario and the recommended procedure involving the Risk Management Program (RMP)*Comp, footprint lengths range up to 40 km. However, the RMP*Comp program provides inconsistent results. Threshold quantities are then calculated using an atmospheric dispersion model and several meteorological and land-use scenarios. In the base scenario (winds at 4.3 m/s, neutral stability, urban conditions, and distances of 100, 250, and 1000 m), distance-based weight thresholds are considerably smaller than current listings for most toxic substances. Distance-based and current thresholds have low correlation (e.g. r=0.34) and large discrepancies (e.g. differences up to three orders of magnitude). Alternative scenarios evaluated for distance-based threshold quantities, which used using stable atmospheres and rural settings further reduce the distance-based weight thresholds and increase discrepancies. Linear relationships are shown between threshold quantities and level of concerns for each scenario and dispersion mode (neutral or dense) that allow simple calculation of threshold quantities. The current thresholds may exclude facilities that could pose significant off-site risks, and the thresholds are inconsistent with the off-site consequence analysis (OCA). Recommendations include revising the threshold quantities that determine covered facilities/processes; modifying RMP*Comp to eliminate errors; establishing threshold quantities using a more rational approach, e.g. based on hazard zones or distances using credible scenarios; and using the same health-based level of concern in both initial screening and subsequent off-site consequence analyses.


Subject(s)
Chemical Industry/standards , Environmental Pollution/statistics & numerical data , Hazardous Substances , Risk Management , Accident Prevention , Guidelines as Topic , Humans
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