Biochemie und Chemie
Refine
Year of publication
Document Type
- Article (72)
- Book (6)
- Doctoral Thesis (1)
- Preprint (1)
- Review (1)
Has Fulltext
- yes (81)
Is part of the Bibliography
- no (81)
Keywords
- environmental tobacco smoke (4)
- particulate matter (4)
- ubiquitin (3)
- Affinity Labeling (2)
- Complex Formation (2)
- Dehydrogenases (2)
- IAPs (2)
- Lactate Dehydrogenase (2)
- XIAP (2)
- additives (2)
Institute
- Medizin (81) (remove)
Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.
The lipid content of skin plays a determinant role in its barrier function with a particularly important role attributed to linoleic acid and its derivatives. Here we explored the consequences of interfering with the soluble epoxide hydrolase (sEH) on skin homeostasis. sEH; which converts fatty acid epoxides generated by cytochrome P450 enzymes to their corresponding diols, was largely restricted to the epidermis which was enriched in sEH-generated diols. Global deletion of the sEH increased levels of epoxides, including the linoleic acid-derived epoxide; 12,13-epoxyoctadecenoic acid (12,13-EpOME), and increased basal keratinocyte proliferation. sEH deletion (sEH-/- mice) resulted in thicker differentiated spinous and corneocyte layers compared to wild-type mice, a hyperkeratosis phenotype that was reproduced in wild-type mice treated with a sEH inhibitor. sEH deletion made the skin sensitive to inflammation and sEH-/- mice developed thicker imiquimod-induced psoriasis plaques than the control group and were more prone to inflammation triggered by mechanical stress with pronounced infiltration and activation of neutrophils as well as vascular leak and increased 12,13-EpOME and leukotriene (LT) B4 levels. Topical treatment of LTB4 antagonist after stripping successfully inhibited inflammation and neutrophil infiltration both in wild type and sEH-/- skin. While 12,13-EpoME had no effect on the trans-endothelial migration of neutrophils, like LTB4, it effectively induced neutrophil adhesion and activation. These observations indicate that while the increased accumulation of neutrophils in sEH-deficient skin could be attributed to the increase in LTB4 levels, both 12,13-EpOME and LTB4 contribute to neutrophil activation. Our observations identify a protective role of the sEH in the skin and should be taken into account when designing future clinical trials with sEH inhibitors.
Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with amino acids modeled in their standard protonation state the structure diverges far from its starting conformation. In comparison, MD simulations performed with pre-determined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results suggest it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to launching any MD simulations. Furthermore, the combined approach of protonation state prediction and MD simulations can provide valuable information on the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions, but also the atomic modeling of density data.
Highlights
• USP32 deubiquitinates the Ragulator complex subunit LAMTOR1 at lysine (K) 20
• LAMTOR1 K20 ubiquitination impairs its binding to the vacuolar H+-ATPase
• USP32 knockout reduces mTORC1 activity and elevates autophagic flux
• Depletion of USP32 in Caenorhabditis elegans inhibits mTOR and induces autophagy
Summary
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain–containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez. Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.
We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.
The Na+-F1F0-ATPase operon ofAcetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunita and b, a gene encoding a 16-kDa proteolipid (subunit c 1), and two genes encoding 8-kDa proteolipids (subunits c 2 andc 3). Because subunits a,b, and c 1 were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a,b, and c 1 are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunitsa, c 2, c 3,b, δ, α, γ, β, and ε. Biochemical and immunological analyses revealed that subunitsc 1, c 2, andc 3 are all part of the c-oligomer, the first of a F1F0-ATPase that contains 8- and 16-kDa proteolipids.
A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
UV-mikrospektrophotometrische Messungen der Nucleinsäuren- und Eiweißkörper-Konzentration sowie der Kern- und Nukleolengröße nach Virusinfektion und unspezifischer Reizung der Chorion-Allantoismembran zeigen, daß es in beiden Fällen zu einer gleich starken Stimulierung des nucleinsäuren- und eiweißkörperbildenden Systems der Zelle kommt. Bei der Infektion mit Vaccinevirus auf das Ektoderm setzt die Reaktion der Membranzellen in der Eklipse ein, nach Infektion mit Newcastle-Disease-Virus fällt der Titeranstieg mit der Zellreaktion zeitlich zusammen.
New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases
(1995)
Reactive coenzyme analogues ω-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of ω-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability at 0 °C. L actate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazonium pentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3α,20β-Hydroxysteroid dehydrogenase reacts at 0 °C with the ethyl homologue and slowly with the propyl compound. The butyl-and pentyl analogues do not inactivate at 0 °C. Tests with 14C -labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2 ′-phospho-adenosine diphosphate, a structural analogue of NADP +, was prepared by condensation of adenosine-2,3-cyclophospho-5′-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2 ′:3′-cyclonucleotide-3′-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehyd rogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP +-dependent dehydrogenases the com pound has been show n to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.
By means of differential thermoanalysis, the miscibility of the main polar tetraether lipid of Thermoplasma acidophilum with two ester lipids, dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol, resp., in the presence of excess water was studied. It is shown that with increasing fraction of tetraether lipid in the mixture, the transition range of dipalmitoyl phosphatidylcholine is broadened and the temperature of the maximum heat flow (Tm) is shifted to lower temperatures; furthermore, the enthaply change (ΔH) of the transition declines. Similar results were obtained with mixtures of tetraether lipid with dipalmitoyl phosphatidylglycerol. It is therefore concluded that the main polar tetraether lipid of Thermoplasma acidophilum , which essentially forms monomolecular layers, is able to form stable common phases with bilayer-forming ester lipids. Miscibility of the tetraether lipid with dipalmitoyl phosphatidylglycerol, which are both monovalent anions at neutral pH, is also observed in the presence of high proton or calcium ion concentrations.
pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pKa in the range 6.8 -8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotina-mide-ribosyl-P2-ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD+ give evidence for the participation of a group in the mitochondrial enzyme with pKa 6.8, deprotonation of which is necessary for detectable association of NAD+. In contrast the fixation of NAD+ to the cytoplasmic enzyme is independent of pH.
A single procedure for the preparation of lactate dehydrogenase (EC 1.1.1.27), the mitochondrial and cytoplasmic forms of malate dehydrogenase (EC 1.1.1.37), adenylate kinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) from pig heart is described. The five enzymes are obtained in preparative amounts in homogenous form with specific activities equal to or higher than those pre viously reported. Some molecular properties of pig heart pyruvate kinase are determined.
Alkylating NAD-Analogs, Glyceraldehyde-3 Phosphate Dehydrogenase, Half-of-the-Sites Reactivity co-(3-Bromoacetylpyridinio)alkyldiphosphoadenosines with alkyl chain lengths of 2 -6 me thylene groups inactivate glyceraldehyde-3 phosphate dehydrogenase from rabbit muscle. Half-of-the-Sites reactivity is observed in each case: The analogs are covalently bound to highly reactive cysteine residues in two of the four subunits. The remaining two subunits still bind N AD and the reactive SH-groups, although modified by SH-reagents of low molecular weight are not labeled by any of the brominated coenzyme models. This behaviour may be explained by the assumption, that the modification of 2 subunits induces structural changes in the neighboured unoccupied subunits which prevent any attack on reactive cysteine residues caused by fixation and orientation of the bromoketo-coenzyme analog when bound to the active center. Structural similarities of the covalently bound coenzyme analogs in the active center and the native ternary GAPDH-NAD-substrate complex suggest that half-of-the-sites reactivity is a natural characteristic of the enzymes catalytic mechanism.
5-Acetyl-4-methyl-1-(β-D-ribofuranosyl) -imidazole-5′-phosphate reacts with diphenylphospho chloridate forming the asymmetrical pyrophosphate ester. This in turn reacts with tri-n-butylammonium phosphate yielding 5-acetyl-4-methyl-imidazole-riboside-5′-diphosphate and with tri-rcbutylammonium pyrophosphate to give the nucleotide triphosphate.
5-Acetyl-4-methyl-imidazole-riboside-5′-pyrophosphate shows in the test with pyruvate kinase a reaction rate three times slower than that of ADP; but the same Km as that of ADP. The ATP analogue is only about 10% as effective as ATP itself in the test with hexokinase, 3-phosphoglycerate kinase and gluconate kinase. Adenylate kinase and NAD+ kinase show no activity when ATP is replaced by the nucleotide-triphosphate-analogue. In presence of ATP the analogue strongly inhibits the reaction of adenylate kinase.
The NAD analogue [3-(3-acetylpyridinio)-propyl] adenosine pyrophosphate forms enzymically inactive complexes with glyceraldehyde-3-phosphate dehydrogenase from yeast and rabbit skeletal muscle. In the latter enzyme four mol of the analogue are bound with equal affinity inhibiting the enzyme in a competitive way: KI = 0.3 mM as compared to the dissociation constant KD=O.6 mм.
The brominated derivative [3- (3-bromoacetylpyridinio) -propyl] adenosine pyrophosphate is covalently bound to both enzymes causing irreversible loss of enzymic activity. Complete inactivation of the enzyme from muscle requires two moles of the analogue per mol of tetramer. The remaining two sites are still able to bind two mol of NAD+ without regain of enzymic activity. In the case of the yeast enzyme four mol of the analogue are bound. Inactivation of the rabbit muscle enzyme is accompanied by the disappearance of two out of four highly reactive sulfhydryl groups; in the yeast enzyme the four active site cysteine residues are still able to react with DTNB1 the reactivity being diminished significantly.
Hybrid formation between the native enzymes from yeast and skeletal muscle is not affected by the modification of the enzyme. Similarly the sedimentation properties of the covalently modified enzyme are indistinguishable from those of the native molecule. This indicates that both the native and the irreversibly inhibited enzyme are identical regarding their quaternary structure.
Studies on the transport of anions and zwitterions of acidic amino acids in Streptomyces hydrogenans
(1983)
n Streptomyces hydrogenans, acidic amino acfds are taken up either as anions by a specific transport system or as zwitterions via a nonspecific one. Variations in the zwitterion concentration caused by changes in pH influence the uptake and exchange diffusion by the nonspecific system. Differences in pH-optima for ʟ-glutamate and ʟ-aspartate transport are due to the different pK2-values of these amino acids. The anion transport by the specific system is accompanied by a short hyperpolarization of the membrane potential followed by a secondary influx of potassium ions into the cells.
The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the β-ᴅ-2′-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectroscopic methods, including 1H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.
Membrane-Phloretin Interaction, Infrared Raman, ESR Spectroscopy The transport inhibitor phloretin was bound to human red cell membrane and the concomitant structural changes were observed by spectroscopic methods. By the spin labeling method a decrease in fluidity of the membrane was found at 1 and 10 |iM concentrations of the reagent. This result was obtained with the 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, and the 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl lipid spin labels. Infrared spectroscopy of modified membranes revealed an intensity increase of the POO~ band at about 1250 cm-1. Moreover, a shift of the peak at 1050 cm -1 to 1100 cm-1 was observed in the presence of phloretin. Raman spectroscopy of the membranes did not contradict the results found with infrared and ESR spectroscopy: In the phloretin modified membrane we observed a lack of the band at 1085 cm-1, which leads to suggest that the POO" and/or C-C regions are less fluid. Changes of the extracted red cell membrane lipids were less characteristic, and the results differed from those found in red cell membrane.
Lactate dehydrogenase from pig heart is inactivated by the NAD+ -analog P1-N6-(4-azidophenylethyl)adenosine-P2-[4-(3-azidopyridinio)butyl]diphosphate (6) upon irradiation with UV light of wavelengths in the range from 300 to 380 nm. The decrease in enzyme activity can be prevented by the addition of NAD+ and oxalate. The modified enzyme shows a reduced binding capacity for its coenzyme as compared to native lactate dehydrogenase. The amount of incorporated coenzyme is deduced from the ribose content of inactivated enzyme. Tryptic digestion of the modified protein and separation of the peptides by HPLC yields 5 ribose-containing fractions. One of them, fraction 6 6 , is split by treatment with nucleotide pyrophosphatase into two subfractions, 63 and 58. Only subfraction 63 contains ribose. Whereas peptide 58 shows a UV absorption spectrum similar to that of 4-(3-aminopyridinio)-butyl phosphate (3). Amino acid analyses of the peptides indicate that the inactivator forms covalent bonds with different parts of the protein: Peptide 63 is characterized by a great portion of hydrophobic amino acids whereas peptide 58 shows a high degree of hydrophilicity.
A new NAD⊕-isomer was prepared, in which the ᴅ-ribose of the adenosine moiety was sub stituted by the enantiomeric ʟ-ribose. As compared to nicotinamide-adenine-dinucleotide (NAD⊕) and NADH the coenzyme isomer (ᴅ,ʟ)-NAD⊕ and its dihydroform (ᴅ,ʟ)-NADH are far less tightly bound to lactate dehydrogenase and alcohol dehydrogenase from horse liver. In the presence of the second substrate (ᴅ,ʟ)-NAD⊕ and (ᴅ,ʟ)-NADH act as hydrogen acceptor and hydrogen donator, respectively, with lactate dehydrogenase and alcohol dehydrogenases from horse liver and yeast. Compared to NAD⊕ and NADH the Michaelis constants are always increased, the catalytic constants (V/Et) were found to be decreased except for the dihydroform reacting with alcohol dehydrogenase from liver.
[4-(3-Bromoacetylpyridinio)-butyl]adenosine pyrophosphate as a structural analog of NAD+ reacts covalently with the sulfhydryl groups of thiopropyl agarose. 10-20 μmol can be bound to 1 ml gel. Stabilization of the insoluble coenzym e is attained by treatment with sodium boro hydride (NaBH4). This complex when applied to column chromatography, allow s the separation of various dehydrogenases as a result of their different complex stability coefficients. Alcohol dehydrogenase from liver, lactate dehydrogenase, and adenylate kinase, which all bind to the ADP-analog residues of the gel matrix, can thus be separated by different salt gradients. Alcohol dehydrogenase from yeast, however, does not form a complex and can easily be eluted from the column with phosphate buffer. Glyceraldehyde-3 phosphate and aldehyde dehydrogenases can be eluted by the addition of NAD+ or NADH to the buffer. The uncharged 1,4-dihydropyridin ring of the reduced coenzyme produces a more stable complex with the dehydrogenases than the oxidized form.
Sulfhydryl Groups, Methylmercury Containing Inactivator, Coenzyme Analogue Nicotinamide-(S-methylmercury-thioinosine) dinucleotide was formed by reaction of nicotin amide-(6-thiopurine) dinucleotide with methylmercury chloride. The compound exhibits coenzyme properties in the test with LDH (Km=1.5 × 10-4 м , Vmax=12500) and LADH (Km=1.7 × 10-4 м, Vmax=27) and inactivates YADH and GAPDH. From incubations with LDH and LADH the mercury containing coenzyme could be regained by column chromatography. The compound seems to be qualified for the X-ray structure analysis of the coenzyme-enzyme complex for some dehyrogenases based on the proportion of the heavy metal.
[ω- (3-Acetylpyridinio) -n-alkyl] adenosine pyrophosphates are coenzyme analogs of NAD⊕. The adenosine pyrophosphate moiety and the 3-acetylpyridine ring of the analogs are connected by n-alkyl chains of different lengths (ethyl -hexyl). The analogs form strong dissociating complexes with lactate dehydrogenase. The complex formation is predominantly achieved by interaction of the ADP moiety with its respective binding domain at the active site.
The redox potentials of the analogs and NAD are of similar magnitude. The coenzyme function of the analogs depends upon the length of the hydrocarbon chain. Lactate dehydrogenase and alcohol dehydrogenases from yeast and horse liver do not catalize hydrogen transfer from their substrates to any other alkyl analog but [4- (3-acetylpyridinio)-n-butyl] adenosine pyrophosphate, aldehyde dehydrogenase from horse liver catalizes hydrogen transfer from acetaldehyde to the pentyl derivative and glyceraldehyde-3-phosphate dehydrogenase catalizes hydrogen transfer to both analogs. In no case, hydrogen transfer from or to one of the 3-acetylpyridine-n-alkyl analogs proceeded with a velocity comparable to NAD or its 3-acetylpyridine analog. The results show that the nicotinamide bound ribose in NAD is involved in the binding and the activation of the coenzyme.
Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30–37 °C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.
The discovery of clustered regularly interspaced short palindromic repeats and their associated proteins (Cas) has revolutionized the field of genome and epigenome editing. A number of new methods have been developed to precisely control the function and activity of Cas proteins, including fusion proteins and small-molecule modulators. Proteolysis-targeting chimeras (PROTACs) represent a new concept using the ubiquitin-proteasome system to degrade a protein of interest, highlighting the significance of chemically induced protein-E3 ligase interaction in drug discovery. Here, we engineered Cas proteins (Cas9, dCas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe (FCPF) amino acid sequence (known as the π-clamp system) and demonstrate that the modified CasFCPF proteins can be (1) labeled in live cells by perfluoroaromatics carrying the fluorescein or (2) degraded by a perfluoroaromatics-functionalized PROTAC (PROTAC-FCPF). A proteome-wide analysis of PROTAC-FCPF-mediated Cas9FCPF protein degradation revealed a high target specificity, suggesting a wide range of applications of perfluoroaromatics-induced proximity in the regulation of stability, activity, and functionality of any FCPF-tagging protein.
Studies over the past decade have revealed that metabolism profoundly influences immune responses. In particular, metabolism causes epigenetic regulation of gene expression, as a growing number of metabolic intermediates are substrates for histone post-translational modifications altering chromatin structure. One of these substrates is acetyl-coenzyme A (CoA), which donates an acetyl group for histone acetylation. Cytosolic acetyl-CoA is also a critical substrate for de novo synthesis of fatty acids and sterols necessary for rapid cellular growth. One of the main enzymes catalyzing cytosolic acetyl-CoA formation is ATP-citrate lyase (ACLY). In addition to its classical function in the provision of acetyl-CoA for de novo lipogenesis, ACLY contributes to epigenetic regulation through histone acetylation, which is increasingly appreciated. In this review we explore the current knowledge of ACLY and acetyl-CoA in mediating innate and adaptive immune responses. We focus on the role of ACLY in supporting de novo lipogenesis in immune cells as well as on its impact on epigenetic alterations. Moreover, we summarize alternative sources of acetyl-CoA and their contribution to metabolic and epigenetic regulation in cells of the immune system.
Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.
Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Children are commonly exposed to second-hand smoke (SHS) in the domestic environment or inside vehicles of smokers. Unfortunately, prenatal tobacco smoke (PTS) exposure is still common, too. SHS is hazardous to the health of smokers and non-smokers, but especially to that of children. SHS and PTS increase the risk for children to develop cancers and can trigger or worsen asthma and allergies, modulate the immune status, and is harmful to lung, heart and blood vessels. Smoking during pregnancy can cause pregnancy complications and poor birth outcomes as well as changes in the development of the foetus. Lately, some of the molecular and genetic mechanisms that cause adverse health effects in children have been identified. In this review, some of the current insights are discussed. In this regard, it has been found in children that SHS and PTS exposure is associated with changes in levels of enzymes, hormones, and expression of genes, micro RNAs, and proteins. PTS and SHS exposure are major elicitors of mechanisms of oxidative stress. Genetic predisposition can compound the health effects of PTS and SHS exposure. Epigenetic effects might influence in utero gene expression and disease susceptibility. Hence, the limitation of domestic and public exposure to SHS as well as PTS exposure has to be in the focus of policymakers and the public in order to save the health of children at an early age. Global substantial smoke-free policies, health communication campaigns, and behavioural interventions are useful and should be mandatory.
Short linear motifs (SLiMs) located in disordered regions of multidomain proteins are important for the organization of protein–protein interaction networks. By dynamic association with their binding partners, SLiMs enable assembly of multiprotein complexes, pivotal for the regulation of various aspects of cell biology in higher organisms. Despite their importance, there is a paucity of molecular tools to study SLiMs of endogenous proteins in live cells. LC3 interacting regions (LIRs), being quintessential for orchestrating diverse stages of autophagy, are a prominent example of SLiMs and mediate binding to the ubiquitin-like LC3/GABARAP family of proteins. The role of LIRs ranges from the posttranslational processing of their binding partners at early stages of autophagy to the binding of selective autophagy receptors (SARs) to the autophagosome. In order to generate tools to study LIRs in cells, we engineered high affinity binders of LIR motifs of three archetypical SARs: OPTN, p62, and NDP52. In an array of in vitro and cellular assays, the engineered binders were shown to have greatly improved affinity and specificity when compared with the endogenous LC3/GABARAP family of proteins, thus providing a unique possibility for modulating LIR interactions in living systems. We exploited these novel tools to study the impact of LIR inhibition on the fitness and the responsiveness to cytarabine treatment of THP-1 cells – a model for studying acute myeloid leukemia (AML). Our results demonstrate that inhibition of LIR of a single autophagy receptor is insufficient to sensitize the cells to cytarabine, while simultaneous inhibition of three LIR motifs in three distinct SARs reduces the IC50 of the chemotherapeutic.
From a global viewpoint, a lot of time is spent within the indoor air compartment of vehicles. A German study on mobility has revealed that, on average, people spend 45 minutes per day inside vehicles. In recent years the number of cars has increased to around 43 million vehicles in private households. This means that more than one car can be used in every household. The ratio has been growing, especially in eastern Germany and rural areas. "Overall and especially outside the cities, the car remains by far number one mode of transport, especially in terms of mileage". Therefore, numerous international studies have addressed different aspects of indoor air hygiene, in the past years. In this paper, meaningful original studies on car indoor air pollution, related to VOCs, COx, PMs, microbials, BFRs, OPFRs, cigarettes, electronic smoking devices, high molecular weight plasticizer, and NOx are summarized in the form of a review. This present review aimed to summarize recently published studies in this important field of environmental medicine and points to the need for further studies with special recommendations for optimizing the interior air hygiene.
The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.
Corneal topometric, aberrometric and biomechanical parameters in mucopolysaccharidosis patients
(2019)
Aims: To report corneal topometric and aberrometric values in mucopolysaccharidosis (MPS) and to investigate their correlation with biomechanical corneal parameters.
Methods: One randomly chosen eye of 20 MPS patients with no to moderate corneal clouding and one eye of 23 healthy controls with comparable age were prospectively included into this study. Corneal surface regularity was assessed by index of surface variance (ISV), -vertical asymmetry (IVA), -height asymmetry (IHA), -height decentration (IHD); keratoconus index (KI), central keratoconus index (CKI) and Zernike indices of anterior and posterior corneal surface using Scheimpflug imaging (Pentacam). Corneal resistance factor (CRF) and corneal hysteresis (CH) were assessed by Ocular Response Analyzer. Statistical analyses were performed using Mann-Whitney-Test and Spearman Correlation Coefficients.
Results: IVA, ISV, IHD, IHA, but not KI and CKI were significantly higher in MPS patients compared to age matched healthy controls. Spherical aberration and asphericity coefficients either at the anterior or at the posterior corneal surface differed significantly between both groups. The grade of the MPS-associated corneal opacity correlated significantly with ISV (rho = 0.52), IVA (rho = 0.54), IHA (rho = 0.57) and IHD (rho = 0.48). Density of the MPS-affected corneas correlated significantly with ISV (rho = 0.52), IVA (rho = 0.72), IHA (rho = 0.57), IHD (rho = 0.69), 3rd order horizontal trefoil aberration at the posterior (rho = 0.62) and anterior surface (rho = 0.48) as well as with CH (rho = 0.55) and CRF (rho = 0.57). Spherical aberration at the back surface correlated with CRF and CH in MPS and in healthy controls.
Conclusions: This is the first study analyzing shape of the corneal surface in MPS patients. Topometric indices of corneal asymmetry are significantly increased and correlate with MPS-related corneal opacity and density. Spherical aberration and asphericity coefficient at the front and at the back corneal surface differ significantly between MPS and healthy controls.
Background: Esophageal cancer (EC) is one of the deadliest cancers worldwide. The contemporary strong increase of the adenocarcinomas in Western countries and the high mortality rates require the intensification of prospective multinational studies.
Methods: Therefore, this global health issue has been chosen for the bibliometric review of the global publication output. As source for meta and citation data, the Web of Science has been used and Density Equalizing Maps were applied for visualization.
Results: 17,387 articles on EC could be identified. The years with publication and citation maxima correspond to the appearance of the most prolific articles. China is the most publishing country, followed by Japan and the USA. Germany and the UK ranked 4th and 5th. The analysis of the ratios articles and socio-economic parameters emphasizes the leading position of the Scandinavian countries and Japan. Here, the high-income countries come out on top. The high incidence regions are mainly represented by Chinese and Japanese research. The association of the publication output and the overall research funding could be shown.
Conclusions: A strengthened international network increasingly consisting of the scientifically best positioned countries as well as more of the high incidence countries worldwide is mandatory for future research. The findings deliver scientists, clinicians and decision makers backgrounds for future decisions all over the world.
Objective: Inhaled particulate matter (PM) in secondhand smoke (SHS) is deleterious for smokers and non-smokers. Different additives in cigarettes might effect the amount of PM. This study aimed to assess the influence of additives on the PM emissions from different cigarette types in SHS.
Design: An experimental study of PM measuring in SHS of cigarettes without exposition of any person.
Method: The concentrations of PM (PM10, PM2.5 and PM1) in SHS of four different types of cigarettes of the brand Lucky Strike, two types with additives (Original Red, Original Blue) and two types without additives (Straight Red, Straight Blue), in comparison to the reference cigarette 3R4F were analysed. An automatic environmental tobacco smoke emitter generated SHS in an enclosed space with a volume of 2.88 m3. PM was measured with a laser aerosol spectrometer (Grimm model 1.109). Afterwards, the measuring values of the four Lucky Strike brands and the reference cigarette were statistically evaluated and visualised.
Results: Lucky Strike Straight Blue, a cigarette type without additives and lower tar amount, showed 10% to 25% lower PM mean values compared with the other tested Lucky Strike products, but 21% (PM1) respectively 27% (PM2.5,PM10) higher mean values than the reference cigarette. The PM mean of all measured smoke-free baseline values (clean air) was 1.6 µg/m³. It increased up to about 1800 µg/m³ for the reference cigarette and up to about 3070 µg/m³ for the Lucky Strike Original Blue.
Conclusions: The findings of this study show the massive increase of PM amount by smoking cigarettes in enclosed spaces and suggest that additives in tobacco products increase the PM amount in SHS. For validation, further comparative studies are necessary focusing on the comparison of the PM concentration of cigarettes with and without additives.
Implications: Due to the exposure to SHS, 890 000 people die each year worldwide. PM in SHS endangers the health of both non-smokers and smokers. This study considers the effect of additives like aromatics and humectant agents in cigarettes on PM in SHS. Do additives in tobacco products increase the amount of PM?
The inhalation of particulate matter (PM) in second-hand smoke (SHS) is hazardous to health of smokers and non-smokers. Tobacco strength (amount of tar, nicotine, and carbon monoxide) and different additives might have an effect on the amount of PM. This study aimed to investigate the influence of tobacco strength or additives on PM. Four cigarette types of the brand Marlboro with different strengths and with or without additives were analyzed in comparison to the 3R4F reference cigarette. SHS was generated by an automatic environmental tobacco smoke emitter (AETSE) in an enclosed space with a volume of 2.88 m³. PM concentrations (PM10, PM2.5, PM1) were measured with a laser aerosol spectrometer followed by statistical analysis. The two strongest Marlboro brands (Red and Red without additives) showed the highest PM concentrations of all tested cigarettes. The measured mean concentrations Cmean of PM10 increased up to 1458 µg/m³ for the Marlboro Red without additives (PM2.5: 1452 µg/m³, PM1: 1263 µg/m³). The similarly strong Marlboro Red showed very similar PM values. The second strongest type Marlboro Gold showed 36% (PM10, PM2.5) and 32% (PM1) lower values, respectively. The “lightest” type Marlboro Silver Blue showed 54% (PM10, PM2.5) or 50% (PM1) lower PM values. The results indicate that the lower the tar, nicotine, and carbon monoxide amounts, as well as the longer the cigarette filter, the lower are the PM levels. An influence of additives could not be determined.
Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.
Air pollution of particulate matter (PM) from traffic emissions has a significant impact on human health. Risk assessments for different traffic participants are often performed on the basis of data from local air quality monitoring stations. Numerous studies demonstrated the limitation of this approach. To assess the risk of PM exposure to a car driver more realistically, we measure the exposure to PM in a car cabin with a mobile aerosol spectrometer in Frankfurt am Main under different settings (local variations, opened versus a closed window) and compare it with data from stationary measurement. A video camera monitored the surroundings for potential PM source detection. In-cabin concentrations peaked at 508 µg m−3 for PM10, 133.9 µg m−3 for PM2.5, and 401.3 µg m−3 for coarse particles, and strongly depended on PM size and PM concentration in ambient air. The concentration of smaller particles showed low fluctuations, but the concentration of coarse particles showed high fluctuations with maximum values on busy roads. Several of these concentration peaks were assigned to the corresponding sources with characteristic particle size distribution profiles. The closure of the car window reduced the exposure to PM, and in particular to coarse particles. The mobile measured PM values differed significantly from stationary PM measures, although good correlations were computed for finer particles. Mobile rather than stationary measurements are essential to assess the risk of PM exposure for car passengers.
Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping. Here we show that fixing loop TMH1-2ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2ND3. We conclude that movement of loop TMH1-2ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.
Background: Immigration has a strong impact on the development of health systems, medicine and science worldwide. Therefore, this article provides a descriptive study on the overall research output.
Methods: Utilizing the scientific database Web of Science, data research was performed. The gathered bibliometric data was analyzed using the established platform NewQIS, a benchmarking system to visualize research quantity and quality indices.
Findings: Between 1900 and 2016 a total of 6763 articles on immigration were retrieved and analyzed. 86 different countries participated in the publications. Quantitatively the United States followed by Canada and Spain were prominent regarding the article numbers. On comparing by additionally taking the population size into account, Israel followed by Sweden and Norway showed the highest performance. The main releasing journals are the Public Health Reports, the Journal of Immigrant and Minority Health and Social Science & Medicine. Over the decades, an increasing number of Public, Environmental & Occupational Health articles can be recognized which finally forms the mainly used subject area.
Conclusion: Considerably increasing scientific work on immigration cannot only be explained by the general increase of scientific work but is also owed to the latest development with increased mobility, worldwide crises and the need of flight and migration. Especially countries with a good economic situation are highly affected by immigrants and prominent in their publication output on immigration, since the countries’ publication effort is connected with the appointed expenditures for research and development. Remarkable numbers of immigrants throughout Europe compel medical professionals to consider neglected diseases, requires the public health system to restructure itself and finally promotes science.
Background: How a dentist works, such as the patterns of movements performed daily, is also largely affected by the workstation Dental tasks are often executed in awkward body positions, thereby causing a very high degree of strain on the corresponding muscles. The objective of this study is to detect those dental tasks, during which awkward postures occur most frequently. The isolated analysis of static postures will examine the duration for which these postures are maintained during the corresponding dental, respectively non-dental, activities.
Methods: 21 (11f/10 m) dentists (age: 40.1 ± 10.4 years) participated in this study. An average dental workday was collected for every subject. To collect kinematic data of all activities, the CUELA system was used. Parallel to the kinematic examination, a detailed computer-based task analysis was conducted. Afterwards, both data sets were synchronized based on the chronological order of the postures assumed in the trunk and the head region. All tasks performed were assigned to the categories "treatment" (I), "office" (II) and "other activities" (III). The angle values of each body region (evaluation parameter) were examined and assessed corresponding to ergonomic standards. Moreover, this study placed a particular focus on static positions, which are held statically for 4 s and longer.
Results: For "treatment" (I), the entire head and trunk area is anteriorly tilted while the back is twisted to the right, in (II) and (III) the back is anteriorly tilted and twisted to the right (non-neutral position). Static positions in (I) last for 4–10s, static postures (approx. 60%) can be observed while in (II) and (III) in the back area static positions for more than 30 s are most common. Moreover, in (II) the back is twisted to the right for more than 60 s in 26.8%.
Conclusion: Awkward positions are a major part of a dentists’ work. This mainly pertains to static positions of the trunk and head in contrast to "office work." These insights facilitate the quantitative description of the dentist profession with regard to the related physical load along with the health hazards to the musculoskeletal system. Moreover, the results allow for a selective extraction of the most unfavorable static body positions that dentists assume for each of the activities performed.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complex membrane protein complexes. The enzyme contributes substantially to oxidative energy-conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. Here, we report the X-ray structure of mitochondrial complex I at 3.6- 3.9 Å resolution describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the ‘deactive’ form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site rendering support for a two-state stabilization-change mechanism of protonpumping.
Post-translational modification of proteins with ubiquitin-like SUMO modifiers is a tightly regulated and highly dynamic process. The SENP family of SUMO-specific isopeptidases comprises six cysteine proteases. They are instrumental in counterbalancing SUMO conjugation, but their regulation is not well understood. We demonstrate that in hypoxic cell extracts, the catalytic activity of SENP family members, in particular SENP1 and SENP3, is inhibited in a rapid and fully reversible process. Comparative mass spectrometry from normoxic and hypoxic cells defines a subset of hypoxia-induced SUMO1 targets, including SUMO ligases RanBP2 and PIAS2, glucose transporter 1, and transcriptional regulators. Among the most strongly induced targets, we identified the transcriptional co-repressor BHLHE40, which controls hypoxic gene expression programs. We provide evidence that SUMOylation of BHLHE40 is reversed by SENP1 and contributes to transcriptional repression of the metabolic master regulator gene PGC-1α. We propose a pathway that connects oxygen-controlled SENP activity to hypoxic reprogramming of metabolism.
Abstract
Indoor air pollution with harmful particulate matter (PM) is mainly caused by cigarette smoke. Super-Slim-Size-Cigarettes (SSL) are considered a less harmful alternative to King-Size-Cigarettes (KSC) due to longer filters and relatively low contents. We ask if “Combined Mainstream and Sidestream Smoke” (CMSS)-associated PM levels of SSL are lower than of KSC and thus are potentially less harmful. PM concentrations in CMSS (PM10, PM2.5, and PM1) are measured from four cigarette types of the brand Vogue, using an “automatic-environmental-tobacco-smoke-emitter” (AETSE) and laser aerosol spectrometry: SSL-BLEUE, -MENTHE, -LILAS and KSC-La Cigarette and -3R4F reference. This analysis shows that SSL MENTHE emitted the highest amount of PM, and KSC-La Cigarette the lowest. 3R4F reference emitted PM in the middle range, exceeding SSL BLEUE and falling slightly below SSL LILAS. It emerged that PM1 constituted the biggest proportion of PM emission. The outcome shows significant type-specific differences for emitted PM concentrations. Our results indicate that SSL are potentially more harmful for passive smokers than the respective KSC. However, this study cannot give precise statements about the general influence of the size of a cigarette on PM. Alarming is that PM1 is responsible for the biggest proportion of PM pollution, since smaller particles cause more harmful effects.
Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.